Brandon:LabNotes/Project1/2015-9-21: Difference between revisions

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*T7R1-Nextera XT V2 TOP was already validated on [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-8-3 Update of THS-seq protocol sequences to use Nextera XT V2 sequences], which adatped from Nextera XT V2 sequences detailed [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/Nextera_XT_V2_sequences_and_outline Nextera XT V2 sequences]]
*T7R1-Nextera XT V2 TOP was already validated on [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-8-3 Update of THS-seq protocol sequences to use Nextera XT V2 sequences], which adatped from Nextera XT V2 sequences detailed [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/Nextera_XT_V2_sequences_and_outline Nextera XT V2 sequences]]
*'''Protocol Changes made to THS-seq with nXTv2 for adaptation to multiplex combinatorial indexing for accessibility'''
**[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-9-24 2015-9-24 Validating changes made to THS-seq protocol for combinatorial sequencing]
***'''[DMF] to high, reduce and test, otherwise all other changes are fine'''
***Does tagmentation work fine in the presence of protease inhibitors, and in 6.4 uL tagmentation reaction?
***comparable IVT yields when using 3 uL cells, 1 uL 4X lysis buffer?
***stopping reation with EDTA 15m at 37C fine?
***pooling and redistributing result in loss?
***using 1/3 beads (36 uL v 54 uL) fine?
***eluting in 6 uL off of SPRI beads to hard?
***1.5 uL taq5X versus 2.4 uL?
***~1/2 IVT reagents work fine (8 v 4.8 uL)?
***after 2x stranded cDNA synthesis, no water addition, only 1.8X beads work fine?
***SPRI afer 2X stranded cDNA synthesis eluted in 10 uL? (run beads on gel and see if any product still stuck?)
***second tagmentation in 16 uL?
***adding 4 uL protease to 16 uL?
***adding 5 uL 5X taq for end fill in?
***PCR with addition only of primers and 1 uL taq5X (same for SYBR green)
**[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-9-28 2015-9-28 Trying exo I to remove sss_nXTv2_prmr and cleaning with DNA clean and concentrator]
***'''trying with ExoI and DNA clean/concentrator, smears to dirty with PCR band, used 10% DMF final reaction for RNA processing tagmentation'''
**[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-9-29 2015-9-29 Trying exo I AGAIN to remove sss_nXTv2_prmr and cleaning with DNA clean and concentrator]
***'''trying with ExoI and DNA clean/concentrator with PCR kapa sybr fast, smears better, band still there'''
**[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-9-30 2015-9-30 NEB_T7_Hiscribe_high_yield_versus_Ambion_Maxiscript_for_IVT_RNA_generation]
***'''USE THIS METHOD NOW FOR RNA PROCESSING AFTER 2X STRANDED SYNTHESIS'''




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  '''Final sequencing ready fragment'''
  '''Final sequencing ready fragment NEW (29 bp index run through)'''
 
nXTv2_i5_index_read (29 bp)----->                                      nXTv2_read1 ----->                nXTv2_i7_index_read (29 bp) ----->
5'- AATGATACGGCGACCACCGAGATCTACAC -3'              5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'        5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                                      3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                          <----- nXTv2_read2
 
'''Final sequencing ready fragment OLD'''
   
   
         nXTv2_i5_index_read ----->                                      nXTv2_read1 ----->                        nXTv2_i7_index_read ----->
         nXTv2_i5_index_read ----->                                      nXTv2_read1 ----->                        nXTv2_i7_index_read ----->
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  '''74 bp long, using truncated barcodes from [http://www.nature.com/nmeth/journal/v11/n2/full/nmeth.2772.html?WT.ec_id=NMETH-201402 Quantitative single-cell RNA-seq with unique molecular identifiers]
  '''74 bp long, using truncated barcodes from [http://www.nature.com/nmeth/journal/v11/n2/full/nmeth.2772.html?WT.ec_id=NMETH-201402 Quantitative single-cell RNA-seq with unique molecular identifiers]
   
   
  num. r5_barcode r5_smpl_sheet name r5_full_sequence_(5'_->_3')
  num. r5_barcode RC name r5_full_sequence_(5'_->_3')
  1 TCTAAT ATTAGA scT7_r5001_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
  1 TCTAAT ATTAGA scT7_r5001_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
  2 ACTCGT ACGAGT scT7_r5002_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
  2 ACTCGT ACGAGT scT7_r5002_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
Line 276: Line 313:
  95 AGAGAA TTCTCT scT7_r5095_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
  95 AGAGAA TTCTCT scT7_r5095_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
  96 ATTTGG CCAAAT scT7_r5096_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATTTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
  96 ATTTGG CCAAAT scT7_r5096_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATTTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
97 CGTGAT ATCACG scT7_r5097_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
98 ACATCG CGATGT scT7_r5098_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACATCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
99 GATCTG CAGATC scT7_r5099_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
100 TCAAGT ACTTGA scT7_r5100_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
101 AAGCTA TAGCTT scT7_r5101_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
102 TGTCTT AAGACA scT7_r5102_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
103 AGGAAG CTTCCT scT7_r5103_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
104 GATGAG CTCATC scT7_r5104_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
105 GCGAAT ATTCGC scT7_r5105_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
106 GCAGTA TACTGC scT7_r5106_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCAGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
107 TCACGA TCGTGA scT7_r5107_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCACGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
108 CGCGTA TACGCG scT7_r5108_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
109 GTTCGT ACGAAC scT7_r5109_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
110 GTGGTG CACCAC scT7_r5110_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
111 CAATTC GAATTG scT7_r5111_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAATTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
112 GTTGCG CGCAAC scT7_r5112_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
113 TCAGTT AACTGA scT7_r5113_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCAGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
114 CCCGAT ATCGGG scT7_r5114_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCCGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
115 TGTAGC GCTACA scT7_r5115_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
116 CTCTGG CCAGAG scT7_r5116_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
117 GGCACT AGTGCC scT7_r5117_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
118 GGATGC GCATCC scT7_r5118_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGATGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
119 GGAGAG CTCTCC scT7_r5119_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGAGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
120 TAACGT ACGTTA scT7_r5120_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAACGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
121 ACACAG CTGTGT scT7_r5121_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACACAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
122 CCGCAC GTGCGG scT7_r5122_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
123 GCGATA TATCGC scT7_r5123_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
124 GGACGT ACGTCC scT7_r5124_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGACGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
125 GTCTGA TCAGAC scT7_r5125_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
126 GAAGGA TCCTTC scT7_r5126_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
127 TCTATC GATAGA scT7_r5127_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
128 ATCTGT ACAGAT scT7_r5128_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATCTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
129 ATAGAG CTCTAT scT7_r5129_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATAGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
130 GCTAAA TTTAGC scT7_r5130_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
131 ACCAGG CCTGGT scT7_r5131_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
132 AAGGAA TTCCTT scT7_r5132_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
133 CACGTC GACGTG scT7_r5133_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCACGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
134 CATAAC GTTATG scT7_r5134_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
135 CCATAT ATATGG scT7_r5135_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCATATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
136 GGCGAC GTCGCC scT7_r5136_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
137 TCGCCA TGGCGA scT7_r5137_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
138 AACACG CGTGTT scT7_r5138_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
139 GCTTGG CCAAGC scT7_r5139_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
140 GGTCTC GAGACC scT7_r5140_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
141 ACCGCA TGCGGT scT7_r5141_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
142 GCTGAT ATCAGC scT7_r5142_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
143 AGTGCT AGCACT scT7_r5143_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
144 CAGGGA TCCCTG scT7_r5144_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
145 CATGCG CGCATG scT7_r5145_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
146 TGCCTA TAGGCA scT7_r5146_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
147 CTATAC GTATAG scT7_r5147_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTATACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
148 CCGAGT ACTCGG scT7_r5148_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
149 ACCTGC GCAGGT scT7_r5149_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
150 CAGGAC GTCCTG scT7_r5150_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
151 GGCTTC GAAGCC scT7_r5151_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
152 GTGCTT AAGCAC scT7_r5152_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
153 TCGAAG CTTCGA scT7_r5153_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
154 TGACTG CAGTCA scT7_r5154_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGACTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
155 GATACA TGTATC scT7_r5155_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
156 TCCTTA TAAGGA scT7_r5156_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
157 CGATTT AAATCG scT7_r5157_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGATTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
158 TTACGG CCGTAA scT7_r5158_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTACGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
159 TTGGAA TTCCAA scT7_r5159_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
160 GTCGAG CTCGAC scT7_r5160_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
161 GGCATG CATGCC scT7_r5161_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
162 GTTTCT AGAAAC scT7_r5162_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
163 ACCAAT ATTGGT scT7_r5163_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
164 TACGGC GCCGTA scT7_r5164_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTACGGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
165 CTGTTG CAACAG scT7_r5165_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
166 GAGAGT ACTCTC scT7_r5166_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
167 TACCCG CGGGTA scT7_r5167_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTACCCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
168 TTCGTA TACGAA scT7_r5168_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
169 TAGCTT AAGCTA scT7_r5169_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
170 GCACTG CAGTGC scT7_r5170_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCACTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
171 ACTATG CATAGT scT7_r5171_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
172 GAACTT AAGTTC scT7_r5172_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAACTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
173 TTTGAG CTCAAA scT7_r5173_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTTGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
174 TAGACC GGTCTA scT7_r5174_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
175 CCTAAG CTTAGG scT7_r5175_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
176 TCCAGA TCTGGA scT7_r5176_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
177 AGGTGA TCACCT scT7_r5177_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
178 CCCATC GATGGG scT7_r5178_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCCATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
179 TTGCAC GTGCAA scT7_r5179_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
180 AGTGAG CTCACT scT7_r5180_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
181 TTTCTC GAGAAA scT7_r5181_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTTCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
182 GCTCTA TAGAGC scT7_r5182_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
183 CTATGT ACATAG scT7_r5183_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTATGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
184 AGCGTT AACGCT scT7_r5184_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
185 AAGACT AGTCTT scT7_r5185_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
186 CGGCTA TAGCCG scT7_r5186_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGGCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
187 TATACG CGTATA scT7_r5187_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTATACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
188 GAACCG CGGTTC scT7_r5188_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAACCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
189 GTACGC GCGTAC scT7_r5189_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTACGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
190 TGGAAC GTTCCA scT7_r5190_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
191 AACAGA TCTGTT scT7_r5191_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
192 AAGCAC GTGCTT scT7_r5192_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
193 CTTCTA TAGAAG scT7_r5193_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
194 ATCGCC GGCGAT scT7_r5194_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATCGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
195 AGCTCT AGAGCT scT7_r5195_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
196 GACGGT ACCGTC scT7_r5196_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGACGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
197 CGACAG CTGTCG scT7_r5197_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGACAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
198 ACATGT ACATGT scT7_r5198_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACATGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
199 ATACGA TCGTAT scT7_r5199_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATACGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
200 ATCCTA TAGGAT scT7_r5200_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATCCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
201 GGAAAC GTTTCC scT7_r5201_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGAAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
202 AGCAAC GTTGCT scT7_r5202_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
203 CTAGTG CACTAG scT7_r5203_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTAGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
204 CCGCTT AAGCGG scT7_r5204_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
205 CTCTAT ATAGAG scT7_r5205_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCTATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
206 GGATCT AGATCC scT7_r5206_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGATCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
207 CACCTG CAGGTG scT7_r5207_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCACCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
208 CATGAA TTCATG scT7_r5208_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
209 CGTGGA TCCACG scT7_r5209_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
210 ATCCAG CTGGAT scT7_r5210_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATCCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
211 CAACAA TTGTTG scT7_r5211_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAACAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
212 CCTGTT AACAGG scT7_r5212_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
213 CTGAGA TCTCAG scT7_r5213_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
214 TCCACT AGTGGA scT7_r5214_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
215 CAAGAG CTCTTG scT7_r5215_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
216 GGTTCC GGAACC scT7_r5216_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
217 AAAGCA TGCTTT scT7_r5217_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAAGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
218 TGCAGT ACTGCA scT7_r5218_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
219 TGTTCT AGAACA scT7_r5219_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
220 TATCGC GCGATA scT7_r5220_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
221 TAAGAC GTCTTA scT7_r5221_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAAGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
222 ACTCTC GAGAGT scT7_r5222_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
223 TCAACG CGTTGA scT7_r5223_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCAACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
224 ACGATT AATCGT scT7_r5224_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACGATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
225 AACTTG CAAGTT scT7_r5225_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
226 AACGTA TACGTT scT7_r5226_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
227 ACCCAC GTGGGT scT7_r5227_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
228 CGGTCT AGACCG scT7_r5228_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGGTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
229 GGTGTG CACACC scT7_r5229_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
230 ACCACC GGTGGT scT7_r5230_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
231 GACTGC GCAGTC scT7_r5231_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGACTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
232 TGTGCG CGCACA scT7_r5232_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
233 CGAGAC GTCTCG scT7_r5233_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
234 GGGATT AATCCC scT7_r5234_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGGATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
235 GTCGTC GACGAC scT7_r5235_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
236 GCATTC GAATGC scT7_r5236_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCATTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
237 TCTGGG CCCAGA scT7_r5237_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTGGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
238 GCCAGT ACTGGC scT7_r5238_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCCAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
239 CCAGTC GACTGG scT7_r5239_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCAGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
240 TTGGCC GGCCAA scT7_r5240_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
241 GTAACA TGTTAC scT7_r5241_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
242 TCTCCT AGGAGA scT7_r5242_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
243 AGATCA TGATCT scT7_r5243_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGATCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
244 CTTTGA TCAAAG scT7_r5244_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
245 TCGCAT ATGCGA scT7_r5245_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
246 CTGTCC GGACAG scT7_r5246_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
247 CGATCC GGATCG scT7_r5247_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGATCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
248 TGGAGG CCTCCA scT7_r5248_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
249 GAATAG CTATTC scT7_r5249_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAATAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
250 CAACGG CCGTTG scT7_r5250_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAACGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
251 GCGGAA TTCCGC scT7_r5251_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
252 GATGGC GCCATC scT7_r5252_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATGGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
253 CCGATG CATCGG scT7_r5253_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
254 GATTTC GAAATC scT7_r5254_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
255 CCAAAC GTTTGG scT7_r5255_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCAAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
256 AGGATC GATCCT scT7_r5256_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
257 CATTCA TGAATG scT7_r5257_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
258 AGATTG CAATCT scT7_r5258_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGATTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
259 CGAAGC GCTTCG scT7_r5259_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
260 TAGGAG CTCCTA scT7_r5260_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
261 CTACCG CGGTAG scT7_r5261_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTACCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
262 AATGGA TCCATT scT7_r5262_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAATGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
263 TTCGAC GTCGAA scT7_r5263_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
264 AACCGG CCGGTT scT7_r5264_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACCGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
265 AAGGCC GGCCTT scT7_r5265_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
266 CGTCCT AGGACG scT7_r5266_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
267 AAGAGC GCTCTT scT7_r5267_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
268 GTGAAA TTTCAC scT7_r5268_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
269 ACGAAC GTTCGT scT7_r5269_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACGAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
270 CAAGGC GCCTTG scT7_r5270_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAGGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
271 ATGTGC GCACAT scT7_r5271_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATGTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
272 CCCTTG CAAGGG scT7_r5272_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCCTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
273 GAGAAG CTTCTC scT7_r5273_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
274 GTAGTT AACTAC scT7_r5274_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
275 TGGTGC GCACCA scT7_r5275_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
276 GACTAT ATAGTC scT7_r5276_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGACTATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
277 CTCGCA TGCGAG scT7_r5277_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
278 TCTTGT ACAAGA scT7_r5278_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
279 AAGCGT ACGCTT scT7_r5279_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
280 TGAGCC GGCTCA scT7_r5280_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGAGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
281 ACGGAG CTCCGT scT7_r5281_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACGGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
282 CAGCCA TGGCTG scT7_r5282_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
283 TATTGG CCAATA scT7_r5283_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTATTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
284 CACTCG CGAGTG scT7_r5284_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCACTCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
285 GCTATT AATAGC scT7_r5285_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
286 CTCCAC GTGGAG scT7_r5286_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
287 GTTAGC GCTAAC scT7_r5287_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
288 AAACCT AGGTTT scT7_r5288_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAACCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
289 CTAGAT ATCTAG scT7_r5289_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTAGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
290 GAAGAT ATCTTC scT7_r5290_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
291 TGGCTC GAGCCA scT7_r5291_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
292 GGTTAT ATAACC scT7_r5292_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTTATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
293 GGAAGA TCTTCC scT7_r5293_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGAAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
294 TACGTG CACGTA scT7_r5294_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTACGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
295 GCTTAC GTAAGC scT7_r5295_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
296 TTCTGC GCAGAA scT7_r5296_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
297 GTGTTA TAACAC scT7_r5297_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
298 CATCGA TCGATG scT7_r5298_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
299 TTCATC GATGAA scT7_r5299_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
300 CCTCTG CAGAGG scT7_r5300_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
301 CCAAGG CCTTGG scT7_r5301_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCAAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
302 AGAGTC GACTCT scT7_r5302_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
303 ACTCAA TTGAGT scT7_r5303_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
304 CTGAAT ATTCAG scT7_r5304_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
305 GTCTAC GTAGAC scT7_r5305_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
306 TCGGAC GTCCGA scT7_r5306_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
307 AGGGAC GTCCCT scT7_r5307_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
308 AGCTAG CTAGCT scT7_r5308_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
309 GTAAGT ACTTAC scT7_r5309_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
310 TGCGGA TCCGCA scT7_r5310_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
311 CAGTAA TTACTG scT7_r5311_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
312 TAGGTC GACCTA scT7_r5312_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
313 TAGGCT AGCCTA scT7_r5313_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
314 CTTACG CGTAAG scT7_r5314_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
315 ACACCC GGGTGT scT7_r5315_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACACCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
316 TGAACA TGTTCA scT7_r5316_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGAACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
317 ATCTCG CGAGAT scT7_r5317_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATCTCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
318 GACCCA TGGGTC scT7_r5318_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGACCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
319 GCCTCA TGAGGC scT7_r5319_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCCTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
320 TACACA TGTGTA scT7_r5320_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTACACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
321 GAGGTA TACCTC scT7_r5321_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
322 CGAATG CATTCG scT7_r5322_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
323 TATCCA TGGATA scT7_r5323_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTATCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
324 CCGACA TGTCGG scT7_r5324_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
325 GATCCC GGGATC scT7_r5325_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATCCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
326 GAGCAA TTGCTC scT7_r5326_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
327 TCCATG CATGGA scT7_r5327_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
328 TGACAT ATGTCA scT7_r5328_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGACATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
329 TTCGGT ACCGAA scT7_r5329_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
330 TCCCGT ACGGGA scT7_r5330_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
331 ATCGAT ATCGAT scT7_r5331_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATCGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
332 CCTTCC GGAAGG scT7_r5332_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
333 CTGCTC GAGCAG scT7_r5333_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
334 GGGTCA TGACCC scT7_r5334_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGGTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
335 GATGCT AGCATC scT7_r5335_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
336 CGCACA TGTGCG scT7_r5336_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
337 TTGTTC GAACAA scT7_r5337_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
338 TGGTTA TAACCA scT7_r5338_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
339 CTGCCT AGGCAG scT7_r5339_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
340 TGCTCA TGAGCA scT7_r5340_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
341 GGTTGA TCAACC scT7_r5341_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
342 AAGTGG CCACTT scT7_r5342_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
343 CTGCAA TTGCAG scT7_r5343_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
344 GCTACC GGTAGC scT7_r5344_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
345 TCTACA TGTAGA scT7_r5345_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
346 CAGTGC GCACTG scT7_r5346_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
347 AGAGGG CCCTCT scT7_r5347_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
348 CTTCGC GCGAAG scT7_r5348_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
349 GGTACG CGTACC scT7_r5349_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
350 TTTACC GGTAAA scT7_r5350_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTTACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
351 CCCTAC GTAGGG scT7_r5351_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCCTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
352 TATCAG CTGATA scT7_r5352_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
353 ATGAGG CCTCAT scT7_r5353_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATGAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
354 ATAGGT ACCTAT scT7_r5354_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATAGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
355 GGACCA TGGTCC scT7_r5355_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGACCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
356 CGTTGC GCAACG scT7_r5356_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
357 TTACCA TGGTAA scT7_r5357_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTACCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
358 GGCCTT AAGGCC scT7_r5358_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
359 TCGGTG CACCGA scT7_r5359_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
360 CAGATC GATCTG scT7_r5360_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
361 GCAAGC GCTTGC scT7_r5361_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCAAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
362 AAGTTC GAACTT scT7_r5362_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
363 GAGTTT AAACTC scT7_r5363_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGTTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
364 AGTCTG CAGACT scT7_r5364_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
365 AAACTC GAGTTT scT7_r5365_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAACTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
366 AACCAT ATGGTT scT7_r5366_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
367 AATTGC GCAATT scT7_r5367_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAATTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
368 AGAAGT ACTTCT scT7_r5368_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
369 AGATAC GTATCT scT7_r5369_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGATACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
370 ATCACA TGTGAT scT7_r5370_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATCACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
371 ATGACC GGTCAT scT7_r5371_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATGACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
372 ATGCCA TGGCAT scT7_r5372_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATGCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
373 ATTGCA TGCAAT scT7_r5373_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATTGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
374 CACACT AGTGTG scT7_r5374_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCACACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
375 CACATA TATGTG scT7_r5375_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCACATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
376 CATATG CATATG scT7_r5376_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
377 CCTCAT ATGAGG scT7_r5377_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
378 CGTATT AATACG scT7_r5378_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
379 CTCAAA TTTGAG scT7_r5379_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
380 CTCTTA TAAGAG scT7_r5380_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
381 GAAATC GATTTC scT7_r5381_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
382 GGTAGT ACTACC scT7_r5382_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
383 GTATTG CAATAC scT7_r5383_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTATTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
384 GTTCAG CTGAAC scT7_r5384_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
    
    
   
   
Line 302: Line 628:
   
   
   
   
   num. i7_barcode i7_name i7_smplsheet i7_full_sequence_(5'_->_3')
   num. i7_barcode i7_long_name i7_smplsheet i7_full_sequence_(5'_->_3')
  1 TCGCCTTA N701_long TAAGGCGA CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG
  1 TCGCCTTA N701_long TAAGGCGA CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG
  2 CTAGTACG N702_long CGTACTAG CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG
  2 CTAGTACG N702_long CGTACTAG CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG
Line 617: Line 943:




===Results===
*total RNA.
*TBU gel after IVT, Guanidine HCl to remove proteins
*qPCR curves
*after adding barcodes, TBE gel
*gel size selection amounts
*gel size selection
*gel size selection validation
===conclusions===
*submitted for sequencing
===multiplex combinatorial cellular indexing adaptation to THS-seq using nXTv2 sequences to assay chromatin accessibility protocol===
*Does tagmentation work fine in the presence of protease inhibitors?
*Using 16% DMF final concentration in solution, any effect on dissociating nuclei? didn't with 10%
*sorting by FACS with DAPI added necessary? just add by dilution?
*using  1/3 beads and 1/2 IVT reagents work fine?
*96 well RNA purification plate for all samples, versus doing beads?
*after 2x stranded cDNA synthesis, no water addition, only 1.8X beads work fine?
*second tagmentation in 20 uL?
*SPRI after second tagmentation eluted in 10 uL? (run beads on gel and see if any product still stuck?)
*reagents etc needed:
**taq5X ~ 1 mL (100)
**clontech MMLV, 1/2 kit, and 1 order of dNTPS (125)
**SPRI beads (~10 mL per run)  (175)
**maxiscript ~1/2 kit per run (150)
**zymo ZR RNA clean and concentrator plate 1 plate, (200)
**qiagen protease, 1/2  (70)
===Before starting protocols===
1. Check if have enough reagents etc for the protocol
*lysis buffer
*tn5059 transposomes
*custom 3' transpson
*IVT reagents
*cells etc
*5X, 2X taq polymerase
*Zymo DNA clean and concentrator
*2. Samples
samples
1. XX
2. XX
===IVT Protocol===
*If need to make more transposome, do first 2 steps. If not goto step 3.
'''Generation of transposomes'''
1. annealing of ME sequence to T7 transposon sequence
a. Make 100 uM stock solution of tranposon and ME only bottom end.
b. Incubate 15 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 2 minutes, then cool to 14C at 0.1 C/s.
c. Oligo's now at 30 uM in 50 uL.
2. Transposome complex generation, run controls!!!
*add the below components into one tube and incubate for 30 minutes at RT
a. Add 1.0 uL of 100% sterile glycerol to tube
b. Add 1.0 uL of annealed transposon to the 100% sterile glycerol and mix well
c. Add 2.0 uL of transposase to well.
*store at -20, is good for a year
3. Prepare samples, lyse cells with lysis buffer
CELL WASHING - (wash cells with PBS)
a. count cells, spin down all cells at 250Xg for 5 minutes
b. wash with 1X PBS, centrifuge 250Xg for 5 minutes, resuspend to 33 cells/uL
c. Count cells again to be sure of cells/uL number
CELL LYSIS
a. dilute Lysis Buffer (LB) to 4X, or as needed (from 10X stock)
b. add 3 uL cells to each well in 96 well plate
c. add 1 uL 4X LB + protease inhibitors to each well, mix briefly
d. transposition ready
LYSIS BUFFER NOTES
*Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.
4. transposition reaction. Add all components and incubate at 37C FOR 30 MINUTES
'''Added in 3 uL cells, then 1 uL 4X LB'''
1.2 uL 5X Custom Tagmentation buffer
4.0 uL lysed cells/pure genomic DNA
1.2 uL T7 transposomes (5X tn5-059, indexed Tsome as listed)
___________
6.4 uL total solution
5. Stop reaction by adding 1 volume (6.5 uL) 40 mM EDTA. Incubate for 15 mins at 37C.
6. Nuclei pooling and recovery
a. Pool all nuclei into one tube
b. Add 8 uL PBS to each well of the 96 well plate, and add to pooled tube
c. Mix pooled tube gently and throughly then aliquot 25 nuclei, 5.25 uL to each well in 96 well plate
        (9600/2016) = 4.76 nuclei/uL;  25/4.76 = 5.25
*sort with FACS? can sort into 10 uL EB buffer.
*In paper: nuclei were first stained with DAPI (Invitrogen) at a final concentration of 3μM and then 25 DAPI-positive nuclei were sorted into each well of a plate with 20μl EB buffer.
7. Bring each well to 10 uL with N-H2O. Add 10 uL 8M guanidine HCl for a total of 20 uL.
**For SPRI beads add 1.8X (36 uL) beads per well and follow beads purification protocol
**'''ELUTE IN 6, leave beads in solution'''
8. Fill in reaction
*Add 1.5 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes.
9. Maxiscript (Ambion) T7 Protocol, IVT
*DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U)
    on ice, but keep 10X transcription buffer at room temp
b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
  X    uL  DNA template (list 1 ug)
  1.2  uL  10X Transcription Buffer
  0.6  uL  10 mM ATP
  0.6  uL  10 mM CTP
  0.6  uL  10 mM GTP
  0.6  uL  10 mM UTP
  1.2  uL  T7 Enzyme Mix
  _______________________
  12.3 uL total
b. Incubate reactions at 37C overnight for ~16-19 hours.
10. Clean with ZR-96 RNA Clean & Concentrator, put solution and all beads on columns.
*using beads could be an option?
*elute samples in 9.0 uL of N-H2O
*quanitate/view on TBU gel, only 1 sample?
'''AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO'''
*Amounts of RNA and water to add for MMLV RT (50 ng RNA)
11. single strand synthesis MMLV RT (Clontech)
*Followed protocol for [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/SMART_MMLV_RT SMART MMLV Reverse Transcriptase]
a. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 11.5 uL with
    Nuclease free H2O
b. heat the mixture to 70C for 3 minutes. Immediately cool on ice.
c. Add the following to the reaction.
    4  uL 5X first strand buffer
    2  uL dNTP mix
    2  uL 100 mM DTT
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ___________________________
    20 uL total
d. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes.
e. Terminate the reaction by heating at 70C for 10 minutes
12. RNase H digestion
*Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction.
a. Add 1 uL of 10X diluted Rnase H to the reaction.
b. Incubate for 20 minutes at 37C.
13. Second strand synthesis: Adding sss_scnXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_scnXTv2 has a high Tm.
a. Add 2.5 uL of 20 uM sss_scnXTv2 to each reaction.
b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice.
c. Add 5.875 uL taq5X to reactions, Incubate at 72C for 8 minutes
14. Clean DNA with AMPURE beads depending. elute in 10 uL
*'''USED AMPURE DNA BEADS AT 1:1.8X RATIO'''
*add 54 uL beads per reaction
*elute on 10 uL
15. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6
3 uL custom tagmentation buffer
X uL cDNA sample
X uL Nuclase free H2O
3 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST)
___________
16 uL total solution
Incubate at 55C for 6 minutes, cool briefly on ice after
16. Protease digestion of transposase, protease inactivation
To each tube, add:
4 uL  Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final [])
Incubate: 50C 10 minutes, 70C 20 minutes
17. Fill in reaction
*Add 5 uL 5X taq polymerase, run at 72C for 3 minutes.
18. PCR addition of barcodes '''USING NEXTERA XT V2 modified i5 (scT7_S5XX) AND i7 (N7XX) SEQUENCES!!!!!'''
Samples:
1. dlkd
2. fdko
KAPA SYBR FAST qPCR mix until saturation, X35 cycles
1  uL Taq5X
2  uL scT7_S5XX index primer, 2 uL F, (scT7_S5XX) '''USED XXX'''
2  uL nXTv2_i7 index primer, 2 uL R, (i7XX_long or i7XX_orig)
1  uL 25X SYBR green
25 uL DNA template (use half RT reaction)
_____________
30 uL
KAPA SYBR cycles:
72C 3m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever
*terminate before curves saturate (usually cycle 6-8)
19. Pool all samples run on 6% PAGE gel, Qubit. Gel Size selection
*gel size select from 220-1000 bp, follow gel size selection protocol
*do not need to include controls.


===Results===
===Results===

Latest revision as of 18:20, 13 April 2016

multiplex combinatorial cellular indexing adaptation to THS-seq using nXTv2 sequence detail and protocol[edit]

  • adapting THS-seq to combinatorial method sequences detail, however will use 3 barcodes since initial transposition only inserts 1 barcode, and barcode will be 6 base pairs to save on transposon length.



  • Protocol Changes made to THS-seq with nXTv2 for adaptation to multiplex combinatorial indexing for accessibility
    • 2015-9-24 Validating changes made to THS-seq protocol for combinatorial sequencing
      • [DMF] to high, reduce and test, otherwise all other changes are fine
      • Does tagmentation work fine in the presence of protease inhibitors, and in 6.4 uL tagmentation reaction?
      • comparable IVT yields when using 3 uL cells, 1 uL 4X lysis buffer?
      • stopping reation with EDTA 15m at 37C fine?
      • pooling and redistributing result in loss?
      • using 1/3 beads (36 uL v 54 uL) fine?
      • eluting in 6 uL off of SPRI beads to hard?
      • 1.5 uL taq5X versus 2.4 uL?
      • ~1/2 IVT reagents work fine (8 v 4.8 uL)?
      • after 2x stranded cDNA synthesis, no water addition, only 1.8X beads work fine?
      • SPRI afer 2X stranded cDNA synthesis eluted in 10 uL? (run beads on gel and see if any product still stuck?)
      • second tagmentation in 16 uL?
      • adding 4 uL protease to 16 uL?
      • adding 5 uL 5X taq for end fill in?
      • PCR with addition only of primers and 1 uL taq5X (same for SYBR green)


Original T7R1-Nextera XT V2 TOP (64 bp)

5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’            (T7-nXTv2_i5-1-top)
     \---/  \-/\---------------------/ \--------------------------------/
     spcr T7 rec.  T7 req. forward         read seq.  \-----------------/
                                                              ME


scT7R1-nXTv2_top (74 bp)

5 basepair spacer removed from 5' end
barcode (r5) and {TCCACGCGC} added in, was new sequence added in on combinatorial method, TCCACGC[r5]GC originally

5'- [AATTAATACGACTCACTATAGGGAGA]{TCCACGCGC}(XXXXXX)TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’            (scT7_r5XXX_i5_top)
     \-/\---------------------/                    \--------------------------------/
   T7 rec.  T7 req. forward                            read seq.  \-----------------/
                                                                           ME


Nextera XT V2 Bottom (19 bp with /5Phos/)
 
5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot)



T7 description:
                                       +1
         [CATGG][AAT][TAATACGACTCACTATA|G|GGAGA]
[end sequence]   rec.   T7 req -->        [front sequence for optimal binding]



After Annealing:

5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’         (scT7_r5XXX_i5_top)
                                                     3'- TCTACACATATTCTCTGTC/5Phos/-5'   (P-T7tspn-bot)

5'- /5Phos/CTGTCTCTTATACACATCT -3'                                                       (P-T7tspn-bot)
       3'- GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5'  (scT7_r5XXX_i5_top)



Fragments created after transposition:

5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 
                                                     3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5'



Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:

5'- NNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'                         5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN -3’
3'- NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5'                         3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNN -5' 



After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)

5'- [AATTAATACGACTCACTATAGGGAGA]TCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3'
3'- [TTAATTATGCTGAGTGATATCCCTCT]AGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNNN)NNNN -5'

5'- NNNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[r5]GCGCGTGGA[TCTCCCTATAGTGAGTCGTATTAATT] -3'
3'- NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCT[AGAGGGATATCACTCAGCATAATTAA] -5'



After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT.
First G in GGG is the +1 site for the beginning of RNA synthesis for T7.
for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator)
All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix"

RNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3'



After single strand cDNA synthesis to form cDNA/RNA hybrid

RNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3'
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5'



After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer.
Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x)

DNA  5'- GGGAGATCCACGCGC -3'                                                      (Tm=57C, anneal at 52C) (sss_scnXTv2)
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5'



Now have double stranded DNA, which should be long.

DNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3'
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5'
Custom Nextera i7 transposome
i7 adapter
       
5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        (nXTv2_i7_top)
               3'- TCTACACATATTCTCTGTC/5Phos/ -5' (P-nXTv2_i7_bot)


5'- /5Phos/CTGTCTCTTATACACATCT -3'                (P-nXTv2_i7_bot)
       3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top)



After custom nextera tagmentation:

DNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN        GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'



After fill in:

DNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'



Amplification (to add i5/i7 barcodes):
for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator)
'All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix"

5'- AATGATACGGCGACCACCGA -3'                             (i5_PCR_ILAdptr, Tm=59C)
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC -3' (scT7_S5XX, comp region Tm=57C, whole~75C)
                            DNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                            cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                            (i7XX_orig, comp region Tm=57C, whole~71C)   3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                            (i7XX_long, comp region Tm=59C, whole~71C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                            (i7_PCR_ILAdptr, Tm=58C)                                           3'- AGCATACGGCAGAAGACGAAC -5'



Final sequencing ready fragment NEW (29 bp index run through)
 
nXTv2_i5_index_read (29 bp)----->                                      nXTv2_read1 ----->                 nXTv2_i7_index_read (29 bp) ----->
5'- AATGATACGGCGACCACCGAGATCTACAC -3'               5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'         5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                                      3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                          <----- nXTv2_read2

 
Final sequencing ready fragment OLD

       nXTv2_i5_index_read ----->                                      nXTv2_read1 ----->                         nXTv2_i7_index_read ----->
5'- AATGATACGGCGACCACCGAGATCTACAC -3'               5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'         5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                    3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC -5'         3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                        <----- r5_index_read                              <----- nXTv2_read2




Sequences to order

scT7_r5XXX_i5_top   Barcoded Transposons
P-T7tspn-bot        5'- /5Phos/CTGTCTCTTATACACATCT -3'
sss_scnXTv2         5'- GGGAGATCCACGCGC -3'
nXTv2_i7_top        5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'
P-nXTv2_i7_bot      5'- /5Phos/CTGTCTCTTATACACATCT -3'

scT7_S5XX           Modified Nextera XT V2 i5 barcode sequences and adaptors
i7XX_long           Extended Nextera XT V2 i7 barcode sequences and adaptors
i5_PCR_ILAdptr      5'- AATGATACGGCGACCACCGA -3'
i7_PCR_ILAdptr      5'- CAAGCAGAAGACGGCATACGA-3'

nXTv2_read1         5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
nXTv2_i7_index_read 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
nXTv2_i5_index_read 5'- AATGATACGGCGACCACCGAGATCTACAC -3' 
r5_index_read       5'- CTGTCTCTTATACACATCTGACGCTGCCGACGA -3'
nXTv2_read2         5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'


Barcoded scT7_r5XXX_i5_top transposons
74 bp long, using truncated barcodes from Quantitative single-cell RNA-seq with unique molecular identifiers

num.	r5_barcode	RC	name	r5_full_sequence_(5'_->_3')
1	TCTAAT	ATTAGA	scT7_r5001_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
2	ACTCGT	ACGAGT	scT7_r5002_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
3	ACGTCA	TGACGT	scT7_r5003_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACGTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
4	AACGAC	GTCGTT	scT7_r5004_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
5	TTAGCG	CGCTAA	scT7_r5005_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTAGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
6	ACAGAC	GTCTGT	scT7_r5006_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACAGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
7	CGTAGG	CCTACG	scT7_r5007_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
8	GAGCTC	GAGCTC	scT7_r5008_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
9	AATACC	GGTATT	scT7_r5009_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAATACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
10	TCACAC	GTGTGA	scT7_r5010_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
11	CTGGTA	TACCAG	scT7_r5011_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
12	GATAGG	CCTATC	scT7_r5012_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
13	TGTCGA	TCGACA	scT7_r5013_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
14	ACGGGT	ACCCGT	scT7_r5014_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACGGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
15	CAGCAT	ATGCTG	scT7_r5015_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
16	TTGACA	TGTCAA	scT7_r5016_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
17	TTCAAG	CTTGAA	scT7_r5017_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
18	GTCCTG	CAGGAC	scT7_r5018_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
19	CACTGA	TCAGTG	scT7_r5019_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCACTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
20	TACAGG	CCTGTA	scT7_r5020_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTACAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
21	ACCTTT	AAAGGT	scT7_r5021_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCTTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
22	CCGTTC	GAACGG	scT7_r5022_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
23	TGTTTG	CAAACA	scT7_r5023_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
24	GTGGCT	AGCCAC	scT7_r5024_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
25	GTACAA	TTGTAC	scT7_r5025_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTACAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
26	CAAAGT	ACTTTG	scT7_r5026_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
27	GCATGA	TCATGC	scT7_r5027_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCATGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
28	CATTAG	CTAATG	scT7_r5028_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
29	CTCCTT	AAGGAG	scT7_r5029_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
30	ACCCGA	TCGGGT	scT7_r5030_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
31	TGCCAC	GTGGCA	scT7_r5031_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
32	TACCAA	TTGGTA	scT7_r5032_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTACCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
33	CGAGGT	ACCTCG	scT7_r5033_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
34	CCACCA	TGGTGG	scT7_r5034_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCACCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
35	TAATCA	TGATTA	scT7_r5035_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAATCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
36	AGTCCA	TGGACT	scT7_r5036_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
37	CTCAGC	GCTGAG	scT7_r5037_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
38	TGGACT	AGTCCA	scT7_r5038_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
39	CAAACC	GGTTTG	scT7_r5039_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
40	ACTCCG	CGGAGT	scT7_r5040_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
41	ACCGTG	CACGGT	scT7_r5041_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
42	AGAACG	CGTTCT	scT7_r5042_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
43	ATTGAC	GTCAAT	scT7_r5043_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATTGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
44	GTATCC	GGATAC	scT7_r5044_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTATCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
45	CGGAAA	TTTCCG	scT7_r5045_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGGAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
46	TTGTGT	ACACAA	scT7_r5046_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
47	GCACAT	ATGTGC	scT7_r5047_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCACATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
48	GTCATT	AATGAC	scT7_r5048_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
49	CCTATA	TATAGG	scT7_r5049_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
50	ACGCTG	CAGCGT	scT7_r5050_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACGCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
51	GATTGT	ACAATC	scT7_r5051_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
52	ATGTAG	CTACAT	scT7_r5052_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATGTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
53	CGCTGT	ACAGCG	scT7_r5053_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
54	AGACTT	AAGTCT	scT7_r5054_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGACTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
55	TGGGCA	TGCCCA	scT7_r5055_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
56	ACTAGA	TCTAGT	scT7_r5056_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACTAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
57	ATGGGA	TCCCAT	scT7_r5057_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATGGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
58	GCGACG	CGTCGC	scT7_r5058_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
59	GGGTAG	CTACCC	scT7_r5059_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGGTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
60	GGTGAA	TTCACC	scT7_r5060_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
61	CATCTT	AAGATG	scT7_r5061_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
62	ATAATC	GATTAT	scT7_r5062_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATAATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
63	TATGGT	ACCATA	scT7_r5063_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTATGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
64	GACTTA	TAAGTC	scT7_r5064_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGACTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
65	TGATTC	GAATCA	scT7_r5065_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGATTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
66	TAGCGA	TCGCTA	scT7_r5066_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
67	AACTCC	GGAGTT	scT7_r5067_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
68	GAAGTG	CACTTC	scT7_r5068_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
69	GAGTAC	GTACTC	scT7_r5069_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
70	CGCAAT	ATTGCG	scT7_r5070_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
71	CTGGAG	CTCCAG	scT7_r5071_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
72	TGGGTT	AACCCA	scT7_r5072_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
73	TCCTAG	CTAGGA	scT7_r5073_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
74	TCATGC	GCATGA	scT7_r5074_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCATGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
75	TTCCCT	AGGGAA	scT7_r5075_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
76	GTAAAG	CTTTAC	scT7_r5076_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
77	ACAACT	AGTTGT	scT7_r5077_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACAACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
78	AGGCAT	ATGCCT	scT7_r5078_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
79	TGTGTC	GACACA	scT7_r5079_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
80	AGCATA	TATGCT	scT7_r5080_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
81	AGACGC	GCGTCT	scT7_r5081_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGACGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
82	CTTGCT	AGCAAG	scT7_r5082_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
83	TCTGCC	GGCAGA	scT7_r5083_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
84	TAGTCG	CGACTA	scT7_r5084_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGTCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
85	TTGCTG	CAGCAA	scT7_r5085_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
86	GACAAC	GTTGTC	scT7_r5086_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGACAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
87	ATTCCC	GGGAAT	scT7_r5087_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATTCCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
88	TTAAGC	GCTTAA	scT7_r5088_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTAAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
89	CGCCTC	GAGGCG	scT7_r5089_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
90	CTTATC	GATAAG	scT7_r5090_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
91	CGCTAA	TTAGCG	scT7_r5091_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
92	AGGTCC	GGACCT	scT7_r5092_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
93	AAGGTG	CACCTT	scT7_r5093_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
94	GTTGTA	TACAAC	scT7_r5094_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
95	AGAGAA	TTCTCT	scT7_r5095_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
96	ATTTGG	CCAAAT	scT7_r5096_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATTTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
97	CGTGAT	ATCACG	scT7_r5097_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
98	ACATCG	CGATGT	scT7_r5098_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACATCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
99	GATCTG	CAGATC	scT7_r5099_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
100	TCAAGT	ACTTGA	scT7_r5100_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
101	AAGCTA	TAGCTT	scT7_r5101_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
102	TGTCTT	AAGACA	scT7_r5102_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
103	AGGAAG	CTTCCT	scT7_r5103_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
104	GATGAG	CTCATC	scT7_r5104_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
105	GCGAAT	ATTCGC	scT7_r5105_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
106	GCAGTA	TACTGC	scT7_r5106_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCAGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
107	TCACGA	TCGTGA	scT7_r5107_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCACGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
108	CGCGTA	TACGCG	scT7_r5108_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
109	GTTCGT	ACGAAC	scT7_r5109_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
110	GTGGTG	CACCAC	scT7_r5110_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
111	CAATTC	GAATTG	scT7_r5111_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAATTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
112	GTTGCG	CGCAAC	scT7_r5112_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
113	TCAGTT	AACTGA	scT7_r5113_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCAGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
114	CCCGAT	ATCGGG	scT7_r5114_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCCGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
115	TGTAGC	GCTACA	scT7_r5115_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
116	CTCTGG	CCAGAG	scT7_r5116_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
117	GGCACT	AGTGCC	scT7_r5117_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
118	GGATGC	GCATCC	scT7_r5118_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGATGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
119	GGAGAG	CTCTCC	scT7_r5119_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGAGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
120	TAACGT	ACGTTA	scT7_r5120_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAACGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
121	ACACAG	CTGTGT	scT7_r5121_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACACAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
122	CCGCAC	GTGCGG	scT7_r5122_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
123	GCGATA	TATCGC	scT7_r5123_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
124	GGACGT	ACGTCC	scT7_r5124_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGACGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
125	GTCTGA	TCAGAC	scT7_r5125_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
126	GAAGGA	TCCTTC	scT7_r5126_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
127	TCTATC	GATAGA	scT7_r5127_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
128	ATCTGT	ACAGAT	scT7_r5128_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATCTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
129	ATAGAG	CTCTAT	scT7_r5129_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATAGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
130	GCTAAA	TTTAGC	scT7_r5130_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
131	ACCAGG	CCTGGT	scT7_r5131_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
132	AAGGAA	TTCCTT	scT7_r5132_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
133	CACGTC	GACGTG	scT7_r5133_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCACGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
134	CATAAC	GTTATG	scT7_r5134_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
135	CCATAT	ATATGG	scT7_r5135_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCATATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
136	GGCGAC	GTCGCC	scT7_r5136_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
137	TCGCCA	TGGCGA	scT7_r5137_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
138	AACACG	CGTGTT	scT7_r5138_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
139	GCTTGG	CCAAGC	scT7_r5139_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
140	GGTCTC	GAGACC	scT7_r5140_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
141	ACCGCA	TGCGGT	scT7_r5141_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
142	GCTGAT	ATCAGC	scT7_r5142_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
143	AGTGCT	AGCACT	scT7_r5143_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
144	CAGGGA	TCCCTG	scT7_r5144_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
145	CATGCG	CGCATG	scT7_r5145_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
146	TGCCTA	TAGGCA	scT7_r5146_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
147	CTATAC	GTATAG	scT7_r5147_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTATACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
148	CCGAGT	ACTCGG	scT7_r5148_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
149	ACCTGC	GCAGGT	scT7_r5149_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
150	CAGGAC	GTCCTG	scT7_r5150_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
151	GGCTTC	GAAGCC	scT7_r5151_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
152	GTGCTT	AAGCAC	scT7_r5152_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
153	TCGAAG	CTTCGA	scT7_r5153_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
154	TGACTG	CAGTCA	scT7_r5154_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGACTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
155	GATACA	TGTATC	scT7_r5155_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
156	TCCTTA	TAAGGA	scT7_r5156_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
157	CGATTT	AAATCG	scT7_r5157_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGATTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
158	TTACGG	CCGTAA	scT7_r5158_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTACGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
159	TTGGAA	TTCCAA	scT7_r5159_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
160	GTCGAG	CTCGAC	scT7_r5160_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
161	GGCATG	CATGCC	scT7_r5161_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
162	GTTTCT	AGAAAC	scT7_r5162_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
163	ACCAAT	ATTGGT	scT7_r5163_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
164	TACGGC	GCCGTA	scT7_r5164_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTACGGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
165	CTGTTG	CAACAG	scT7_r5165_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
166	GAGAGT	ACTCTC	scT7_r5166_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
167	TACCCG	CGGGTA	scT7_r5167_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTACCCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
168	TTCGTA	TACGAA	scT7_r5168_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
169	TAGCTT	AAGCTA	scT7_r5169_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
170	GCACTG	CAGTGC	scT7_r5170_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCACTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
171	ACTATG	CATAGT	scT7_r5171_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACTATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
172	GAACTT	AAGTTC	scT7_r5172_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAACTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
173	TTTGAG	CTCAAA	scT7_r5173_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTTGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
174	TAGACC	GGTCTA	scT7_r5174_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
175	CCTAAG	CTTAGG	scT7_r5175_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
176	TCCAGA	TCTGGA	scT7_r5176_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
177	AGGTGA	TCACCT	scT7_r5177_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
178	CCCATC	GATGGG	scT7_r5178_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCCATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
179	TTGCAC	GTGCAA	scT7_r5179_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
180	AGTGAG	CTCACT	scT7_r5180_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
181	TTTCTC	GAGAAA	scT7_r5181_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTTCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
182	GCTCTA	TAGAGC	scT7_r5182_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
183	CTATGT	ACATAG	scT7_r5183_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTATGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
184	AGCGTT	AACGCT	scT7_r5184_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
185	AAGACT	AGTCTT	scT7_r5185_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
186	CGGCTA	TAGCCG	scT7_r5186_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGGCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
187	TATACG	CGTATA	scT7_r5187_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTATACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
188	GAACCG	CGGTTC	scT7_r5188_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAACCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
189	GTACGC	GCGTAC	scT7_r5189_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTACGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
190	TGGAAC	GTTCCA	scT7_r5190_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
191	AACAGA	TCTGTT	scT7_r5191_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
192	AAGCAC	GTGCTT	scT7_r5192_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
193	CTTCTA	TAGAAG	scT7_r5193_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
194	ATCGCC	GGCGAT	scT7_r5194_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATCGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
195	AGCTCT	AGAGCT	scT7_r5195_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
196	GACGGT	ACCGTC	scT7_r5196_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGACGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
197	CGACAG	CTGTCG	scT7_r5197_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGACAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
198	ACATGT	ACATGT	scT7_r5198_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACATGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
199	ATACGA	TCGTAT	scT7_r5199_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATACGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
200	ATCCTA	TAGGAT	scT7_r5200_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATCCTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
201	GGAAAC	GTTTCC	scT7_r5201_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGAAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
202	AGCAAC	GTTGCT	scT7_r5202_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
203	CTAGTG	CACTAG	scT7_r5203_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTAGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
204	CCGCTT	AAGCGG	scT7_r5204_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
205	CTCTAT	ATAGAG	scT7_r5205_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCTATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
206	GGATCT	AGATCC	scT7_r5206_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGATCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
207	CACCTG	CAGGTG	scT7_r5207_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCACCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
208	CATGAA	TTCATG	scT7_r5208_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
209	CGTGGA	TCCACG	scT7_r5209_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
210	ATCCAG	CTGGAT	scT7_r5210_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATCCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
211	CAACAA	TTGTTG	scT7_r5211_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAACAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
212	CCTGTT	AACAGG	scT7_r5212_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
213	CTGAGA	TCTCAG	scT7_r5213_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
214	TCCACT	AGTGGA	scT7_r5214_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
215	CAAGAG	CTCTTG	scT7_r5215_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
216	GGTTCC	GGAACC	scT7_r5216_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
217	AAAGCA	TGCTTT	scT7_r5217_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAAGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
218	TGCAGT	ACTGCA	scT7_r5218_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
219	TGTTCT	AGAACA	scT7_r5219_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
220	TATCGC	GCGATA	scT7_r5220_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
221	TAAGAC	GTCTTA	scT7_r5221_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAAGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
222	ACTCTC	GAGAGT	scT7_r5222_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
223	TCAACG	CGTTGA	scT7_r5223_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCAACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
224	ACGATT	AATCGT	scT7_r5224_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACGATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
225	AACTTG	CAAGTT	scT7_r5225_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
226	AACGTA	TACGTT	scT7_r5226_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
227	ACCCAC	GTGGGT	scT7_r5227_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
228	CGGTCT	AGACCG	scT7_r5228_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGGTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
229	GGTGTG	CACACC	scT7_r5229_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
230	ACCACC	GGTGGT	scT7_r5230_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
231	GACTGC	GCAGTC	scT7_r5231_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGACTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
232	TGTGCG	CGCACA	scT7_r5232_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
233	CGAGAC	GTCTCG	scT7_r5233_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
234	GGGATT	AATCCC	scT7_r5234_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGGATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
235	GTCGTC	GACGAC	scT7_r5235_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
236	GCATTC	GAATGC	scT7_r5236_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCATTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
237	TCTGGG	CCCAGA	scT7_r5237_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTGGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
238	GCCAGT	ACTGGC	scT7_r5238_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCCAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
239	CCAGTC	GACTGG	scT7_r5239_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCAGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
240	TTGGCC	GGCCAA	scT7_r5240_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
241	GTAACA	TGTTAC	scT7_r5241_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
242	TCTCCT	AGGAGA	scT7_r5242_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
243	AGATCA	TGATCT	scT7_r5243_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGATCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
244	CTTTGA	TCAAAG	scT7_r5244_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
245	TCGCAT	ATGCGA	scT7_r5245_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
246	CTGTCC	GGACAG	scT7_r5246_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
247	CGATCC	GGATCG	scT7_r5247_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGATCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
248	TGGAGG	CCTCCA	scT7_r5248_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
249	GAATAG	CTATTC	scT7_r5249_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAATAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
250	CAACGG	CCGTTG	scT7_r5250_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAACGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
251	GCGGAA	TTCCGC	scT7_r5251_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
252	GATGGC	GCCATC	scT7_r5252_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATGGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
253	CCGATG	CATCGG	scT7_r5253_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
254	GATTTC	GAAATC	scT7_r5254_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
255	CCAAAC	GTTTGG	scT7_r5255_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCAAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
256	AGGATC	GATCCT	scT7_r5256_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
257	CATTCA	TGAATG	scT7_r5257_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
258	AGATTG	CAATCT	scT7_r5258_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGATTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
259	CGAAGC	GCTTCG	scT7_r5259_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
260	TAGGAG	CTCCTA	scT7_r5260_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
261	CTACCG	CGGTAG	scT7_r5261_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTACCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
262	AATGGA	TCCATT	scT7_r5262_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAATGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
263	TTCGAC	GTCGAA	scT7_r5263_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
264	AACCGG	CCGGTT	scT7_r5264_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACCGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
265	AAGGCC	GGCCTT	scT7_r5265_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
266	CGTCCT	AGGACG	scT7_r5266_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
267	AAGAGC	GCTCTT	scT7_r5267_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
268	GTGAAA	TTTCAC	scT7_r5268_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
269	ACGAAC	GTTCGT	scT7_r5269_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACGAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
270	CAAGGC	GCCTTG	scT7_r5270_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAGGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
271	ATGTGC	GCACAT	scT7_r5271_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATGTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
272	CCCTTG	CAAGGG	scT7_r5272_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCCTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
273	GAGAAG	CTTCTC	scT7_r5273_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
274	GTAGTT	AACTAC	scT7_r5274_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
275	TGGTGC	GCACCA	scT7_r5275_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
276	GACTAT	ATAGTC	scT7_r5276_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGACTATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
277	CTCGCA	TGCGAG	scT7_r5277_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
278	TCTTGT	ACAAGA	scT7_r5278_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
279	AAGCGT	ACGCTT	scT7_r5279_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
280	TGAGCC	GGCTCA	scT7_r5280_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGAGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
281	ACGGAG	CTCCGT	scT7_r5281_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACGGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
282	CAGCCA	TGGCTG	scT7_r5282_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
283	TATTGG	CCAATA	scT7_r5283_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTATTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
284	CACTCG	CGAGTG	scT7_r5284_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCACTCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
285	GCTATT	AATAGC	scT7_r5285_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
286	CTCCAC	GTGGAG	scT7_r5286_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
287	GTTAGC	GCTAAC	scT7_r5287_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
288	AAACCT	AGGTTT	scT7_r5288_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAACCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
289	CTAGAT	ATCTAG	scT7_r5289_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTAGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
290	GAAGAT	ATCTTC	scT7_r5290_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
291	TGGCTC	GAGCCA	scT7_r5291_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
292	GGTTAT	ATAACC	scT7_r5292_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTTATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
293	GGAAGA	TCTTCC	scT7_r5293_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGAAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
294	TACGTG	CACGTA	scT7_r5294_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTACGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
295	GCTTAC	GTAAGC	scT7_r5295_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
296	TTCTGC	GCAGAA	scT7_r5296_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
297	GTGTTA	TAACAC	scT7_r5297_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
298	CATCGA	TCGATG	scT7_r5298_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
299	TTCATC	GATGAA	scT7_r5299_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
300	CCTCTG	CAGAGG	scT7_r5300_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
301	CCAAGG	CCTTGG	scT7_r5301_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCAAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
302	AGAGTC	GACTCT	scT7_r5302_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
303	ACTCAA	TTGAGT	scT7_r5303_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
304	CTGAAT	ATTCAG	scT7_r5304_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
305	GTCTAC	GTAGAC	scT7_r5305_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
306	TCGGAC	GTCCGA	scT7_r5306_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
307	AGGGAC	GTCCCT	scT7_r5307_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
308	AGCTAG	CTAGCT	scT7_r5308_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
309	GTAAGT	ACTTAC	scT7_r5309_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
310	TGCGGA	TCCGCA	scT7_r5310_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
311	CAGTAA	TTACTG	scT7_r5311_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
312	TAGGTC	GACCTA	scT7_r5312_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
313	TAGGCT	AGCCTA	scT7_r5313_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
314	CTTACG	CGTAAG	scT7_r5314_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
315	ACACCC	GGGTGT	scT7_r5315_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACACCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
316	TGAACA	TGTTCA	scT7_r5316_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGAACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
317	ATCTCG	CGAGAT	scT7_r5317_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATCTCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
318	GACCCA	TGGGTC	scT7_r5318_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGACCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
319	GCCTCA	TGAGGC	scT7_r5319_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCCTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
320	TACACA	TGTGTA	scT7_r5320_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTACACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
321	GAGGTA	TACCTC	scT7_r5321_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
322	CGAATG	CATTCG	scT7_r5322_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
323	TATCCA	TGGATA	scT7_r5323_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTATCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
324	CCGACA	TGTCGG	scT7_r5324_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
325	GATCCC	GGGATC	scT7_r5325_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATCCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
326	GAGCAA	TTGCTC	scT7_r5326_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
327	TCCATG	CATGGA	scT7_r5327_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
328	TGACAT	ATGTCA	scT7_r5328_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGACATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
329	TTCGGT	ACCGAA	scT7_r5329_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
330	TCCCGT	ACGGGA	scT7_r5330_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
331	ATCGAT	ATCGAT	scT7_r5331_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATCGATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
332	CCTTCC	GGAAGG	scT7_r5332_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
333	CTGCTC	GAGCAG	scT7_r5333_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
334	GGGTCA	TGACCC	scT7_r5334_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGGTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
335	GATGCT	AGCATC	scT7_r5335_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
336	CGCACA	TGTGCG	scT7_r5336_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
337	TTGTTC	GAACAA	scT7_r5337_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
338	TGGTTA	TAACCA	scT7_r5338_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
339	CTGCCT	AGGCAG	scT7_r5339_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
340	TGCTCA	TGAGCA	scT7_r5340_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
341	GGTTGA	TCAACC	scT7_r5341_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
342	AAGTGG	CCACTT	scT7_r5342_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
343	CTGCAA	TTGCAG	scT7_r5343_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
344	GCTACC	GGTAGC	scT7_r5344_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCTACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
345	TCTACA	TGTAGA	scT7_r5345_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
346	CAGTGC	GCACTG	scT7_r5346_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
347	AGAGGG	CCCTCT	scT7_r5347_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
348	CTTCGC	GCGAAG	scT7_r5348_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
349	GGTACG	CGTACC	scT7_r5349_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
350	TTTACC	GGTAAA	scT7_r5350_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTTACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
351	CCCTAC	GTAGGG	scT7_r5351_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCCTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
352	TATCAG	CTGATA	scT7_r5352_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
353	ATGAGG	CCTCAT	scT7_r5353_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATGAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
354	ATAGGT	ACCTAT	scT7_r5354_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATAGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
355	GGACCA	TGGTCC	scT7_r5355_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGACCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
356	CGTTGC	GCAACG	scT7_r5356_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
357	TTACCA	TGGTAA	scT7_r5357_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTACCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
358	GGCCTT	AAGGCC	scT7_r5358_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGCCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
359	TCGGTG	CACCGA	scT7_r5359_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCGGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
360	CAGATC	GATCTG	scT7_r5360_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
361	GCAAGC	GCTTGC	scT7_r5361_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCAAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
362	AAGTTC	GAACTT	scT7_r5362_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
363	GAGTTT	AAACTC	scT7_r5363_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGTTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
364	AGTCTG	CAGACT	scT7_r5364_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
365	AAACTC	GAGTTT	scT7_r5365_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAACTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
366	AACCAT	ATGGTT	scT7_r5366_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
367	AATTGC	GCAATT	scT7_r5367_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAATTGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
368	AGAAGT	ACTTCT	scT7_r5368_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
369	AGATAC	GTATCT	scT7_r5369_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGATACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
370	ATCACA	TGTGAT	scT7_r5370_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATCACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
371	ATGACC	GGTCAT	scT7_r5371_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATGACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
372	ATGCCA	TGGCAT	scT7_r5372_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATGCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
373	ATTGCA	TGCAAT	scT7_r5373_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATTGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
374	CACACT	AGTGTG	scT7_r5374_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCACACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
375	CACATA	TATGTG	scT7_r5375_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCACATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
376	CATATG	CATATG	scT7_r5376_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATATGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
377	CCTCAT	ATGAGG	scT7_r5377_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
378	CGTATT	AATACG	scT7_r5378_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
379	CTCAAA	TTTGAG	scT7_r5379_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
380	CTCTTA	TAAGAG	scT7_r5380_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
381	GAAATC	GATTTC	scT7_r5381_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
382	GGTAGT	ACTACC	scT7_r5382_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
383	GTATTG	CAATAC	scT7_r5383_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTATTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
384	GTTCAG	CTGAAC	scT7_r5384_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG



Modified Nextera XT V2 i5 barcode sequences and adaptors
same i7 adaptors/barcodes are used as in original Nextera XT V2 system or 2 bp longer ones can be used as listed below

num.	i5_barcode	i5_smplsheet	scT7_i5_name	scT7_i5_full_sequence_(5'_->_3')
1	CTCTCTAT	CTCTCTAT	scT7_S502	AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCCACGCGC
2	TATCCTCT	TATCCTCT	scT7_S503	AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCCACGCGC
3	GTAAGGAG	GTAAGGAG	scT7_S505	AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCCACGCGC
4	ACTGCATA	ACTGCATA	scT7_S506	AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCCACGCGC
5	AAGGAGTA	AAGGAGTA	scT7_S507	AATGATACGGCGACCACCGAGATCTACACAAGGAGTAGGGAGATCCACGCGC
6	CTAAGCCT	CTAAGCCT	scT7_S508	AATGATACGGCGACCACCGAGATCTACACCTAAGCCTGGGAGATCCACGCGC
7	CGTCTAAT	CGTCTAAT	scT7_S510	AATGATACGGCGACCACCGAGATCTACACCGTCTAATGGGAGATCCACGCGC
8	TCTCTCCG	TCTCTCCG	scT7_S511	AATGATACGGCGACCACCGAGATCTACACTCTCTCCGGGGAGATCCACGCGC
9	TCGACTAG	TCGACTAG	scT7_S513	AATGATACGGCGACCACCGAGATCTACACTCGACTAGGGGAGATCCACGCGC
10	TTCTAGCT	TTCTAGCT	scT7_S515	AATGATACGGCGACCACCGAGATCTACACTTCTAGCTGGGAGATCCACGCGC
11	CCTAGAGT	CCTAGAGT	scT7_S516	AATGATACGGCGACCACCGAGATCTACACCCTAGAGTGGGAGATCCACGCGC
12	GCGTAAGA	GCGTAAGA	scT7_S517	AATGATACGGCGACCACCGAGATCTACACGCGTAAGAGGGAGATCCACGCGC
13	CTATTAAG	CTATTAAG	scT7_S518	AATGATACGGCGACCACCGAGATCTACACCTATTAAGGGGAGATCCACGCGC
14	AAGGCTAT	AAGGCTAT	scT7_S520	AATGATACGGCGACCACCGAGATCTACACAAGGCTATGGGAGATCCACGCGC
15	GAGCCTTA	GAGCCTTA	scT7_S521	AATGATACGGCGACCACCGAGATCTACACGAGCCTTAGGGAGATCCACGCGC
16	TTATGCGA	TTATGCGA	scT7_S522	AATGATACGGCGACCACCGAGATCTACACTTATGCGAGGGAGATCCACGCGC


 num.	i7_barcode	i7_long_name	i7_smplsheet	i7_full_sequence_(5'_->_3')
1	TCGCCTTA	N701_long	TAAGGCGA	CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG
2	CTAGTACG	N702_long	CGTACTAG	CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG
3	TTCTGCCT	N703_long	AGGCAGAA	CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG
4	GCTCAGGA	N704_long	TCCTGAGC	CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG
5	AGGAGTCC	N705_long	GGACTCCT	CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAG
6	CATGCCTA	N706_long	TAGGCATG	CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAG
7	GTAGAGAG	N707_long	CTCTCTAC	CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGGAG
8	CAGCCTCG	N710_long	CGAGGCTG	CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGGAG
9	TGCCTCTT	N711_long	AAGAGGCA	CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGGAG
10	TCCTCTAC	N712_long	GTAGAGGA	CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGGAG
11	TCATGAGC	N714_long	GCTCATGA	CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGGAG
12	CCTGAGAT	N715_long	ATCTCAGG	CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGGAG
13	TAGCGAGT	N716_long	ACTCGCTA	CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGGAG
14	GTAGCTCC	N718_long	GGAGCTAC	CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGGAG
15	TACTACGC	N719_long	GCGTAGTA	CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGGAG
16	AGGCTCCG	N720_long	CGGAGCCT	CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGGAG
17	GCAGCGTA	N721_long	TACGCTGC	CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGGAG
18	CTGCGCAT	N722_long	ATGCGCAG	CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGGAG
19	GAGCGCTA	N723_long	TAGCGCTC	CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGGAG
20	CGCTCAGT	N724_long	ACTGAGCG	CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGGAG
21	GTCTTAGG	N726_long	CCTAAGAC	CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGGAG
22	ACTGATCG	N727_long	CGATCAGT	CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGGAG
23	TAGCTGCA	N728_long	TGCAGCTA	CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGGAG
24	GACGTCGA	N729_long	TCGACGTC	CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGGAG

 original Nextera XT V2 i5/i7 barcode sequences and primers

num.	i5_barcode	i5_name	i5_smplsheet	i5_full_sequence_(5'_->_3')
1	CTCTCTAT	S502	CTCTCTAT	AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC
2	TATCCTCT	S503	TATCCTCT	AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC
3	GTAAGGAG	S505	GTAAGGAG	AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC
4	ACTGCATA	S506	ACTGCATA	AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC
5	AAGGAGTA	S507	AAGGAGTA	AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC
6	CTAAGCCT	S508	CTAAGCCT	AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC
7	CGTCTAAT	S510	CGTCTAAT	AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC
8	TCTCTCCG	S511	TCTCTCCG	AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC
9	TCGACTAG	S513	TCGACTAG	AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC
10	TTCTAGCT	S515	TTCTAGCT	AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC
11	CCTAGAGT	S516	CCTAGAGT	AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC
12	GCGTAAGA	S517	GCGTAAGA	AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC
13	CTATTAAG	S518	CTATTAAG	AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC
14	AAGGCTAT	S520	AAGGCTAT	AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTC
15	GAGCCTTA	S521	GAGCCTTA	AATGATACGGCGACCACCGAGATCTACACGAGCCTTATCGTCGGCAGCGTC
16	TTATGCGA	S522	TTATGCGA	AATGATACGGCGACCACCGAGATCTACACTTATGCGATCGTCGGCAGCGTC
				
				
num.	i7_barcode	i7_name	i7_smplsheet	i7_full_sequence_(5'_->_3')
1	TCGCCTTA	N701	TAAGGCGA	CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG
2	CTAGTACG	N702	CGTACTAG	CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG
3	TTCTGCCT	N703	AGGCAGAA	CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG
4	GCTCAGGA	N704	TCCTGAGC	CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG
5	AGGAGTCC	N705	GGACTCCT	CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG
6	CATGCCTA	N706	TAGGCATG	CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG
7	GTAGAGAG	N707	CTCTCTAC	CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG
8	CAGCCTCG	N710	CGAGGCTG	CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG
9	TGCCTCTT	N711	AAGAGGCA	CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG
10	TCCTCTAC	N712	GTAGAGGA	CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG
11	TCATGAGC	N714	GCTCATGA	CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG
12	CCTGAGAT	N715	ATCTCAGG	CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG
13	TAGCGAGT	N716	ACTCGCTA	CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG
14	GTAGCTCC	N718	GGAGCTAC	CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG
15	TACTACGC	N719	GCGTAGTA	CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG
16	AGGCTCCG	N720	CGGAGCCT	CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG
17	GCAGCGTA	N721	TACGCTGC	CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG
18	CTGCGCAT	N722	ATGCGCAG	CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG
19	GAGCGCTA	N723	TAGCGCTC	CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG
20	CGCTCAGT	N724	ACTGAGCG	CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG
21	GTCTTAGG	N726	CCTAAGAC	CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG
22	ACTGATCG	N727	CGATCAGT	CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGG
23	TAGCTGCA	N728	TGCAGCTA	CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGG
24	GACGTCGA	N729	TCGACGTC	CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGG



Validation of THS-seq barcoded transposons using Nextera XT v2 sequences for multiplex combinatorial indexing[edit]

  • designed scT7_r5XXX_i5_top transposon so it can be used used with 6 base pair barcodes. 96 barcoded transposons made. Will first test with tn5059 transposomes from Trina for validation the transposon is working and that fragments can be sequenced on MiSeq/HiSeq. Then will proceed to order the rest of the transposons and use with combinatorial sequencing with THS-seq for single cells.


  • There is slight modification to MiSeq protocol, custom primers will need to be used and Read2 will instead be a 6 basepair index reading using r5_index_read. Standard Nextera XT v2 oligos i5 index, i7 index and read1 are used for sequencing.


Before starting protocols[edit]

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • tn5059 transposomes
  • custom 3' transpson
  • IVT reagents
  • cells etc
  • 5X, 2X taq polymerase
  • Zymo DNA clean and concentrator


  • 2. Samples
samples
1. scT7_i502-i701, scT7_r5001_i5_top, 5X tn5-059, 500 cells rep1
2. scT7_i503-i701, scT7_r5001_i5_top, 5X tn5-059, 500 cells rep2
3. scT7_i505-i701, scT7_r5002_i5_top, 5X tn5-059, 500 cells rep1
4. scT7_i502-i702, scT7_r5002_i5_top, 5X tn5-059, 500 cells rep2
5. scT7_i503-i702, scT7_r5001_i5_top, 5X tn5-059, 6 ng pure DNA
6. scT7_i505-i702, scT7_r5001_i5_top, 5X tn5-059, NTC

IVT Protocol[edit]

  • If need to make more transposome, do first 2 steps. If not goto step 3.

Generation of transposomes

1. annealing of ME sequence to T7 transposon sequence

a. Make 100 uM stock solution of tranposon and ME only bottom end.
b. Incubate 15 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 2 minutes, then cool to 14C at 0.1 C/s.
c. Oligo's now at 30 uM in 50 uL.


2. Transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 30 minutes at RT
a. Add 1.0 uL of 100% sterile glycerol to tube
b. Add 1.0 uL of annealed transposon to the 100% sterile glycerol and mix well
c. Add 2.0 uL of transposase to well.
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer

CELL WASHING - (wash cells with PBS)

a. count cells, spin down all cells at 250Xg for 5 minutes
b. wash with 1X PBS, centrifuge 250Xg for 5 minutes, resuspend to 1000 cells/uL
c. Count cells again to be sure of cells/uL amount.

CELL LYSIS

a. dilute Lysis Buffer (LB) to 2X, or as needed (from 10X stock)
b. add 1 uL LB to sample tubes 
c. add 1 uL cells to sample tubes with LB in it, mix briefly (mineral oil optional)
d. transposition ready


LYSIS BUFFER NOTES

  • Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.


4. transposition reaction. Add all components and incubate at 37C FOR 30 MINUTES

1.0 uL 5X Custom Tagmentation buffer
2.0 uL lysed cells/pure genomic DNA
1.0 uL T7 transposomes (5X tn5-059, indexed Tsome as listed)
1.0 uL N-H2O
___________
5.0 uL total solution


5. Bring to 15 uL with N-H2O. Add 15 uL 8M guanidine HCl for a total of 30 uL.

    • For SPRI beads add 1.8X (54 uL) beads per sample. and follow beads protocol
    • ELUTE IN 9.6, leave beads in solution


6. Fill in reaction

  • Add 2.4 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes.


7. Maxiscript (Ambion) T7 Protocol, IVT

  • DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) 
   on ice, but keep 10X transcription buffer at room temp

b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
  X   uL   DNA template (list 1 ug)
  2   uL   10X Transcription Buffer
  1   uL   10 mM ATP
  1   uL   10 mM CTP
  1   uL   10 mM GTP
  1   uL   10 mM UTP
  2   uL   T7 Enzyme Mix
 _______________________
  20 uL total 


b. Incubate reactions at 37C overnight for ~16-19 hours. (>10 uM limiting nucleotide)


8. Clean with Zymo RNA clean and concentrator, put solution and all beads on column.

  • elute samples in 10 uL of N-H2O
  • quanitate/view on TBU gel.



AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO

sample list
1. dkljd
2. ldkf

  • Amounts of RNA and water to add for MMLV RT (50 ng RNA)


9. single strand synthesis MMLV RT (Clontech)


a. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 11.5 uL with
   Nuclease free H2O

b. heat the mixture to 70C for 3 minutes.  Immediately cool on ice.

c. Add the following to the reaction.
   4  uL 5X first strand buffer
   2  uL dNTP mix
   2  uL 100 mM DTT
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ___________________________
   20 uL total

d. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes.

e. Terminate the reaction by heating at 70C for 10 minutes


10. RNase H digestion

  • Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction.

a. Add 1 uL of 10X diluted Rnase H to the reaction.

b. Incubate for 20 minutes at 37C.


11. Second strand synthesis: Adding sss_scnXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_scnXTv2 has a high Tm.

a. Add 2.5 uL of 20 uM sss_scnXTv2 to each reaction.

b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice.

c. Add 5.875 uL taq5X to reactions, Incubate at 72C for 8 minutes


12. Clean DNA with AMPURE beads depending. elute in 20 uL and concentrate.

  • USED AMPURE DNA BEADS AT 1:1.8X RATIO
  • add 45 uL N-H2O
  • add 135 uL beads per reaction
  • elute on 20 uL


13. After elution in 20 uL, use vacuum centrifuge commentator to concentrate sample size to ~4-5 uL per sample.

  • Also ~4-5 uL per sample is preferred for transposition, which has always been performed in low volume solutions.

RAN FOR 16 MINUTES


14. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6

1 uL custom tagmentation buffer
X uL cDNA sample
X uL Nuclase free H2O
1 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST)
___________
5 uL total solution

Incubate at 55C for 6 minutes, cool briefly on ice after


15. Protease digestion of transposase, protease inactivation

To each tube, add:
1 uL  Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final [])
Incubate: 50C 10 minutes, 70C 20 minutes


16. Fill in reaction

  • Add 6 uL 2X taq polymerase, run at 72C for 3 minutes.


17. PCR addition of barcodes USING NEXTERA XT V2 modified i5 (scT7_S5XX) AND i7 (N7XX) SEQUENCES!!!!!

Samples:
1. dlkd
2. fdko
KAPA SYBR FAST qPCR mix until saturation, X35 cycles

9  uL Taq2X
2  uL scT7_S5XX index primer, 2 uL F, (scT7_S5XX) USED XXX
2  uL nXTv2_i7 index primer, 2 uL R, (i7XX_long or i7XX_orig)
4  uL H2O
1  uL 25X SYBR green
12 uL DNA template (use half RT reaction)
_____________
30 uL

KAPA SYBR cycles:
72C 3m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever

  • terminate before curves saturate (usually cycle 6-8)


18. Gel Size selection

  • gel size select from 220-1000 bp, follow gel size selection protocol
  • do not need to include controls.



Results[edit]

  • total RNA.


  • TBU gel after IVT, Guanidine HCl to remove proteins


  • qPCR curves


  • after adding barcodes, TBE gel


  • gel size selection amounts


  • gel size selection


  • gel size selection validation


conclusions[edit]

  • submitted for sequencing