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 -----> | ||
Line 179: | Line 216: | ||
'''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 | 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 | 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 |
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.
- T7R1-Nextera XT V2 TOP was already validated on Update of THS-seq protocol sequences to use Nextera XT V2 sequences, which adatped from Nextera XT V2 sequences detailed Nextera XT V2 sequences]
- 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)
- 2015-9-24 Validating changes made to THS-seq protocol for combinatorial sequencing
- 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
- 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
- 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
- 2015-9-28 Trying exo I to remove sss_nXTv2_prmr and cleaning with DNA clean and concentrator
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)
- Followed protocol for 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
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