Brandon:LabNotes/Project1/2015-8-3: Difference between revisions

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*[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-7-1 2015-7-1 THS miseq troubleshooting R4]
*[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-7-1 2015-7-1 THS miseq troubleshooting R4]
*[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-7-14 2015-7-14 THS miseq troubleshooting R5]
*[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-7-14 2015-7-14 THS miseq troubleshooting R5]
*[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-7-24 2015-7-24 THS miseq troubleshooting, HiSeq (4/1/2015) Samples that worked, didn't work on MiSeq]




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*Thus will redesign THS-seq using Nextera XT V2 sequencing/index/flowcell/amplification primers.
*Thus will redesign THS-seq using [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/Nextera_XT_V2_sequences_and_outline Nextera XT V2 sequencing/index/flowcell/amplification primers.]






===Sequences information, Using T7-nxtv2_i5-1-top and T7-nxtv2_i5-2-top===
===Sequences information, Using T7-nXTv2_i5-1-top and T7-nXTv2_i5-2-top===
   
   
  Sequences:
  Sequences:
Line 32: Line 33:
       spcr      T7 forward    required spcr  read sequence  spcr      ME  
       spcr      T7 forward    required spcr  read sequence  spcr      ME  
   
   
  '''NEXTERA XT V2 R1 TOP (64 bp)'''
  '''T7R1-NEXTERA XT V2 TOP (64 bp)'''
  5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’            (T7-nxtv2_i5-1-top)
  5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’            (T7-nXTv2_i5-1-top)
       \---/  \-/\---------------------/ \--------------------------------/
       \---/  \-/\---------------------/ \--------------------------------/
       spcr T7 rec.  T7 req. forward        read seq.  \-----------------/
       spcr T7 rec.  T7 req. forward        read seq.  \-----------------/
                                                               ME   
                                                               ME   
                                                                                    
                                                                                    
  '''NEXTERA XT V2 R1 TOP + EXTRA (69 bp)'''
  '''T7R1-NEXTERA XT V2 TOP + EXTRA (69 bp)'''
  5'- [CATGG][AATTAATACGACTCACTATAGGGAGA][CTCCT][TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’    (T7-nxtv2_i5-2-top)     
  5'- [CATGG][AATTAATACGACTCACTATAGGGAGA][CTCCT][TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’    (T7-nXTv2_i5-2-top)     
       \---/  \-/\---------------------/  \---/  \--------------------------------/
       \---/  \-/\---------------------/  \---/  \--------------------------------/
       spcr T7 rec.  T7 req. forward      spcr      read seq.  \-----------------/
       spcr T7 rec.  T7 req. forward      spcr      read seq.  \-----------------/
                                                                       ME   
                                                                       ME   
   
   
'''NEXTERA XT V2 BOTTOM (19 bp with /5Phos/)'''
5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot)
   
   
  5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot)
  '''T7 description:'''
  T7 description:
                                         +1
                                         +1
           [CATGG][AAT][TAATACGACTCACTATA|G|GGAGA]
           [CATGG][AAT][TAATACGACTCACTATA|G|GGAGA]
Line 54: Line 55:
   
   
   
   
  After Annealing:
  '''After Annealing:'''
   
   
         5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’      (T7-nxtv2-1-top)
         5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’      (T7-nXTv2-1-top)
                                                     3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot)
                                                     3'- TCTACACATATTCTCTGTC/5Phos/-5' (P-T7tspn-bot)
   
   
   
   
  5'- [phos] CTGTCTCTTATACACATCT -3'
  5'- /5Phos/CTGTCTCTTATACACATCT -3'
       3’-  GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’
       3’-  GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’
   
   
   
   
  Fragments created after transposition:
  '''Fragments created after transposition:'''
   
   
  5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNN--------- 5P/CTGTCTCTTATACACATCT -3'
  5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNN--------- 5P/CTGTCTCTTATACACATCT -3'
Line 70: Line 71:
   
   
   
   
  Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:
  '''Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:'''
   
   
  5'- NNN--------- 5P/CTGTCTCTTATACACATCT -3'          5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNNNN -3'
  5'- NNN--------- 5P/CTGTCTCTTATACACATCT -3'          5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNNNN -3'
Line 76: Line 77:
   
   
   
   
  After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)
  '''After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)'''
   
   
  5'- CATGG[AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3'
  5'- CATGG[AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN -3'
  3'- GTACC[TTAATTATGCTGAGTGATATCCCTCT]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNN)NNN -5'
  3'- GTACC[TTAATTATGCTGAGTGATATCCCTCT]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNNN)NNN -5'
   
   
  5'- NNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[TCTCCCTATAGTGAGTCGTATTAATT]CCATG -3'
  5'- NNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[TCTCCCTATAGTGAGTCGTATTAATT]CCATG -3'
Line 86: Line 87:
   
   
   
   
  After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT.
  '''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.
  '''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)
  '''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’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3'
  RNA 5’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3'
Line 94: Line 96:
   
   
   
   
  After single strand cDNA synthesis to form cDNA/RNA hybrid
  '''After single strand cDNA synthesis to form cDNA/RNA hybrid'''
   
   
  RNA  5’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'  
  RNA  5’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'  
Line 100: Line 102:
   
   
   
   
  After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer.
  '''After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer.'''
  Extenstion with BST or klenow exo- or DNAP1 or Taq2x
  '''Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x)'''
   
   
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' (Tm=77) (sss_nxtv2_prmr)
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'                 (Tm=70, anneal@64C) (P-sss_nXTv2_prmr)
  cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
  cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
   
   
   
   
  Now have double stranded DNA, which should be long.
  '''Now have double stranded DNA, which should be long.'''
   
   
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN-3' (Tm=77) (sss_nxtv2_prmr)
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
  cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
  cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


  Custom Nextera i7 transposome
  '''Custom Nextera i7 transposome'''
     i7 adapter
     i7 adapter
          
          
     5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        (nxtv2_i7_top)
     5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        (nXTv2_i7_top)
                   3'- TCTACACATATTCTCTGTC/5Phos/ -5' (nxtv2_i7_bot)
                   3'- TCTACACATATTCTCTGTC/5Phos/ -5' (P-nXTv2_i7_bot)
   
   
   
   
     5'- /5Phos/CTGTCTCTTATACACATCT -3'                (nxtv2_i7_bot)
     5'- /5Phos/CTGTCTCTTATACACATCT -3'                (P-nXTv2_i7_bot)
           3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nxtv2_i7_top)
           3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top)
             
   
   
'''After custom nextera tagmentation:'''
   
   
  After custom nextera tagmentation:
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNN--------- 5P/CTGTCTCTTATACACATCT -3'
cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN    GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
   
   
DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3'
cDNA  3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
   
   
   
   
'''After fill in:'''
   
   
  After fill in:
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
   
   
DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
cDNA  3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -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"'''
   
   
Amplification (barcode and ILA adaptor addition):
5’- [AATGATACGGCGACCACCGA]GGG]AGATCCTCCCTCGCGCCATCAGAG    T7-top2-PCR-iaf3 (comp part (Tm=77), all (Tm=85C)
                 
    5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAG    T7-top2-PCR-iaf2 (comp part (Tm=73), all (Tm=83C))
                          X X
    5’- [AATGATACGGCGACCACCGA]GATCTCTCCCTCGCGCCATCAGAGAT  T7-top2-PCR-iaf (original) (comp region 70C) (whole Tm=83C)
                          X X XXXXX
                DNA /5Phos/GGG]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                cDNA  3'- CCC]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (custom NXTA barcodes) (Tm=86C)
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                                                        3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)
   
   
  Read Primers:
  5'- AATGATACGGCGACCACCGA -3'                                  (i5_PCR_ILAdptr, Tm=59C)
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGC -3'    (i5XX_T7-1_shrt, comp region Tm=60C, whole=76C)
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5XX_T7-1_long, comp region Tm=65C, whole=77C)
      5’- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5XX_orig, comp region Tm=56C, whole=75C)
                                      XXXXXX
                      DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                      cDNA        3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                            (i7XX_orig, comp region Tm=57C, whole=73C)  3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i7XX_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i7_PCR_ILAdptr, Tm=58C)                                          3'- AGCATACGGCAGAAGACGAAC -5'
'''PCR barcode addition primers:'''
'''Using i5_shrt or i5_long and i7_orig or i7_long, test to validate'''
    
    
Using T7-top2-PCR-iaf2 for amp.
   
   
                                          (Nextera_R1)
'''Final sequecing ready fragment'''
                          5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
                                        (T7tspn-Read1)                                   (Nextera_Ind index read primer)
        nXTv2_i5_index_read ----->                        nXTv2_read1 ----->                      nXTv2_i7_index_read ----->
                          5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'     5'- CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG -3'
5'- AATGATACGGCGACCACCGAGATCTACAC -3'  5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'      5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
  5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG[XXXXXX]ATCTCGTATGCCGTCTTCTGCTTG -3'
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
  3'- [TTACTATGCCGCTGGTGGCT]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                      3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' 
                                                                                          <----- nXTv2_read2
 
sequences to order:
T7-nXTv2_i5-1-top  5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
T7-nXTv2_i5-2-top  5'- CATGGAATTAATACGACTCACTATAGGGAGACTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
P-T7tspn-bot        5'- /5Phos/CTGTCTCTTATACACATCT -3'
P-sss_nXTv2_prmr    5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
nXTv2_i7_top        5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'       
P-nXTv2_i7_bot      5'- /5Phos/CTGTCTCTTATACACATCT -3'
i5_PCR_ILAdptr      5'- AATGATACGGCGACCACCGA -3'
i7_PCR_ILAdptr      5'- CAAGCAGAAGACGGCATACGA -3'
i502_T7-1_shrt      5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC -3'
i503_T7-1_shrt      5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC -3'
i505_T7-1_shrt      5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC -3'
i506_T7-1_shrt      5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC -3'
i502_T7-1_long      5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGCGTC -3'
i503_T7-1_long      5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGCGTC -3'
i505_T7-1_long      5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGCGTC -3'
i506_T7-1_long      5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGCGTC -3'
i701_long          5'- CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG -3'
i702_long          5'- CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG -3'
i703_long          5'- CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG -3'
i704_long          5'- CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG -3'
sequencing primers used:
nXTv2_i5_index      5'- AATGATACGGCGACCACCGAGATCTACAC -3'
nXTv2_i7_index      5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
nXTv2_read1        5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
nXTv2_read2        5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'
 
'''Modified Nextera XT V2 i5 barcode sequences and adaptors'''
num. i5_barcode i5_name i5_smplsheet i5_full_sequence_(5'_->_3')
1 CTCTCTAT S502_T7-1_shrt CTCTCTAT AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC
2 TATCCTCT S503_T7-1_shrt TATCCTCT AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC
3 GTAAGGAG S505_T7-1_shrt GTAAGGAG AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC
4 ACTGCATA S506_T7-1_shrt ACTGCATA AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC
5 AAGGAGTA S507_T7-1_shrt AAGGAGTA AATGATACGGCGACCACCGAGATCTACACAAGGAGTAGGGAGATCGTCGGCAGC
6 CTAAGCCT S508_T7-1_shrt CTAAGCCT AATGATACGGCGACCACCGAGATCTACACCTAAGCCTGGGAGATCGTCGGCAGC
7 CGTCTAAT S510_T7-1_shrt CGTCTAAT AATGATACGGCGACCACCGAGATCTACACCGTCTAATGGGAGATCGTCGGCAGC
8 TCTCTCCG S511_T7-1_shrt TCTCTCCG AATGATACGGCGACCACCGAGATCTACACTCTCTCCGGGGAGATCGTCGGCAGC
9 TCGACTAG S513_T7-1_shrt TCGACTAG AATGATACGGCGACCACCGAGATCTACACTCGACTAGGGGAGATCGTCGGCAGC
10 TTCTAGCT S515_T7-1_shrt TTCTAGCT AATGATACGGCGACCACCGAGATCTACACTTCTAGCTGGGAGATCGTCGGCAGC
11 CCTAGAGT S516_T7-1_shrt CCTAGAGT AATGATACGGCGACCACCGAGATCTACACCCTAGAGTGGGAGATCGTCGGCAGC
12 GCGTAAGA S517_T7-1_shrt GCGTAAGA AATGATACGGCGACCACCGAGATCTACACGCGTAAGAGGGAGATCGTCGGCAGC
13 CTATTAAG S518_T7-1_shrt CTATTAAG AATGATACGGCGACCACCGAGATCTACACCTATTAAGGGGAGATCGTCGGCAGC
14 AAGGCTAT S520_T7-1_shrt AAGGCTAT AATGATACGGCGACCACCGAGATCTACACAAGGCTATGGGAGATCGTCGGCAGC
15 GAGCCTTA S521_T7-1_shrt GAGCCTTA AATGATACGGCGACCACCGAGATCTACACGAGCCTTAGGGAGATCGTCGGCAGC
16 TTATGCGA S522_T7-1_shrt TTATGCGA AATGATACGGCGACCACCGAGATCTACACTTATGCGAGGGAGATCGTCGGCAGC
 
'''original Nextera XT V2 i5/i7 barcode sequencers and primers'''
num. i5_barcode i5_name i5_smplsheet i5_full_sequence
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
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
 
 
 
 
'''UPDATE 8/27/2015'''
 
[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-8-27 8/27/2015 T7-nXTv2_i5-1 testing]
 
===Validation of THS-seq update to Nextera XT V2 sequences===
 
 
*received tn5059 transposomes T7-nXTv2_i5-1 and nXTv2_i7 from Trina, will run through protocol and MiSeq testing for validation
 
 
===Before starting protocols===
 
1. Check if have enough reagents etc for the protocol
*lysis buffer
*tn5059 transposomes
*custom 3' transpson
*IVT reagents
*cells etc
*5X, 2X taq polymerase
*Zymo DNA clean and concentrator
 
 
*2. Samples
samples
1. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1
2. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2
3. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
4. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, NTC
 
 
===IVT Protocol===
 
*If need to make more transposome, do first 2 steps. If not goto step 3.
 
'''Generation of transposomes'''
 
1. annealing of ME sequence to T7 transposon sequence
a. Make 100 uM stock solution of tranposon and ME only bottom end.
b. Incubate 15 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 2 minutes, then cool to 14C at 0.1 C/s.
c. Oligo's now at 30 uM in 50 uL.
 
 
2. transposome complex generation, run controls!!!
*add the below components into one tube and incubate for 20 minutes at RT
a. Add 0.5 uL of 100% sterile glycerol to tube
b. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well
c. Add 1.0 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, T7-nXTv2_i5-1)
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 with Qubit or on TBU gel.
 
 
 
 
'''AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO'''
 
sample list
21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1
22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2
23. i503_shrt-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
24. i503_shrt-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
25. i505_shrt-i701_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
26. i505_shrt-i702_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
27. i506_long-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
28. i506_long-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
29. i502_long-i703_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
30. i502_long-i704_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
31. i503_shrt-i703_long, 8/27/2015, 4. T7-nXTv2_i5-1, 5X tn5-059, NTC
 
*Amounts of RNA and water to add for MMLV RT (50 ng RNA)
 
 
9. single strand synthesis MMLV RT (Clontech)
 
*Followed protocol for [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/SMART_MMLV_RT SMART MMLV Reverse Transcriptase]
 
20 uL reaction
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 fo 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 P-sss_nXTv2_prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since P-sss_nXTv2_prmr has a high Tm.
 
a. Add 2.5 uL of 20 uM P-sss_nXTv2_prmr 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 concentreate.
 
*'''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 vaccum 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 i5 AND i7 SEQUENCES!!!!!'''
 
Samples:
samples processed during [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-9-8 RNA processing for rest ofjohn chang T cell samples, first cell division, day7 Tsle and Tmp, day 90 memory, AND nXTv2 THS-seq update RNA processing]
21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1
22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2
23. i503_shrt-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
24. i503_shrt-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
25. i505_shrt-i701_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
26. i505_shrt-i702_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
27. i506_long-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
28. i506_long-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
29. i502_long-i703_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
30. i502_long-i704_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
31. i503_shrt-i703_long, 8/27/2015, 4. T7-nXTv2_i5-1, 5X tn5-059, NTC
 
KAPA SYBR FAST qPCR mix until saturation, X35 cycles
9  uL Taq2X
2  uL nXTv2_i5 index primer, 2 uL F, (i5XX_T7-1_shrt, OR long) '''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, 59C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever
55C for comp ends for taq2X
'''HIGH BACKGROUND WITH 59C, NEXT TIME RUN AT 63C'''
*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===
 
 
*total RNA.
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''5406.090161'''
| align="center" style="background:#f0f0f0;"|'''5'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| ||3730.570111||2.5||in 10 uL
|-
| 1. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1||17614.20052||23.21540477||232.1540477
|-
| 2. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2||16680.7305||21.82259836||218.2259836
|-
| 3. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA||142250.0542||209.1813288||2091.813288
|-
| 4. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, NTC||708.0800211||-2.009779054||-20.09779054
|-
|
|}
 
 
 
*TBU gel after IVT, Guanidine HCl to remove proteins
*yields from 500 cells approx. half that seen before, will try PAGE purified transposon instead, and v2 version.
[[File:ZhangLab 2 2015-08-28 11hr 27min-labeled.jpg|600px]]
 
 
 
*qPCR curves
*do not gain much info from efficiency based on PCR curves for short versus long primers. seems 502 works better.
[[File:2015-09-09 nXTv2 validation, primers testing.bmp|600px]]
 
 
 
 
*after adding barcodes, TBE gel
*high amount of primer dimers, maybe since ran at 59C for annealing instead of 63C. next time will try 63C.
[[File:ZhangLab 2 2015-09-09 18hr 14min-labeled.jpg|600px]]
 
*gel size selection amounts
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''6007.810179'''
| align="center" style="background:#f0f0f0;"|'''2.5'''
| align="center" style="background:#f0f0f0;"|'''for 150 ng'''
| align="center" style="background:#f0f0f0;"|'''X2'''
|-
| 21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1||23864.16071||23.11343865||6.48973103||12.97946206
|-
| 22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2||20633.37061||19.38380233||7.738419813||15.47683963
|-
|
|}
 
*gel size selection
[[File:ZhangLab 2 2015-09-10 14hr 03min-labeled.jpg|600px]]
 
 
*gel size selection validation
[[File:ZhangLab 2 2015-09-10 18hr 23min-labeled.jpg|600px]]
 
===conclusions===
 
*submitted for sequencing
 
 
*read stats:
BS-THS-nXTv2-valdation-i502-i701-Sep08_S35_L001_R1_00_trim.fastq
Total Reads: 2668419
Mapped Reads: 2443127
Clonal Reads: 587086
Total Unique Reads: 1856041
91.7% mapping rate
27.1% clonals rate
 
*libraries nXTv2 transposome validated.
*thus will use from now on for accessibility assay.
[[File:NXTv2-validation.png|1000px]]

Latest revision as of 20:38, 10 December 2015

Update of THS-seq protocol sequences to use Nextera XT V2 sequences[edit]



  • Called illumina and they said it is either sequencer not being cleaned right, or concentrations are off, or sequences in library generation protocol are not compatible. Considering currently old nextera sequences are being used from 5 years ago, and all current systems illumina sequencing systems do not use them, then maybe that is the problem.



Sequences information, Using T7-nXTv2_i5-1-top and T7-nXTv2_i5-2-top[edit]

Sequences:
ORIGINAL T7tspn-top2: (69 bp)
5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top2)
      spcr      T7 forward    required spcr   read sequence  spcr       ME 

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  
                                                                                 
T7R1-NEXTERA XT V2 TOP + EXTRA (69 bp)
5'- [CATGG][AATTAATACGACTCACTATAGGGAGA][CTCCT][TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’    (T7-nXTv2_i5-2-top)    
     \---/  \-/\---------------------/  \---/  \--------------------------------/
     spcr T7 rec.  T7 req. forward      spcr      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'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’       (T7-nXTv2-1-top)
                                                    3'- TCTACACATATTCTCTGTC/5Phos/-5' (P-T7tspn-bot)


5'- /5Phos/CTGTCTCTTATACACATCT -3'
      3’-  GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’


Fragments created after transposition:

5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNN--------- 5P/CTGTCTCTTATACACATCT -3'
                                             3'- TCTACACATATTCTCTGTC/5P ---------NNNNNNNNNN    GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’


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

5'- NNN--------- 5P/CTGTCTCTTATACACATCT -3'           5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNNNN -3'
3'- NNNNNNNNNNNN    GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’           3'- TCTACACATATTCTCTGTC/5P ---------NNN -5'


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

5'- CATGG[AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN -3'
3'- GTACC[TTAATTATGCTGAGTGATATCCCTCT]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNNN)NNN -5'

5'- NNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[TCTCCCTATAGTGAGTCGTATTAATT]CCATG -3'
3'- NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[AGAGGGATATCACTCAGCATAATTAA]GGTAC -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’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3'



After single strand cDNA synthesis to form cDNA/RNA hybrid

RNA  5’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' 
cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -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’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'                 (Tm=70, anneal@64C) (P-sss_nXTv2_prmr)
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


Now have double stranded DNA, which should be long.

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -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’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNN--------- 5P/CTGTCTCTTATACACATCT -3'
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN    GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'



After fill in:

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN 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]GGGAGATCGTCGGCAGC -3'    (i5XX_T7-1_shrt, comp region Tm=60C, whole=76C)
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5XX_T7-1_long, comp region Tm=65C, whole=77C)
      5’- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5XX_orig, comp region Tm=56C, whole=75C)
                                     XXXXXX
                     DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                     cDNA        3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                            (i7XX_orig, comp region Tm=57C, whole=73C)   3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i7XX_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i7_PCR_ILAdptr, Tm=58C)                                           3'- AGCATACGGCAGAAGACGAAC -5'

PCR barcode addition primers:
Using i5_shrt or i5_long and i7_orig or i7_long, test to validate
 

Final sequecing ready fragment

       nXTv2_i5_index_read ----->                         nXTv2_read1 ----->                      nXTv2_i7_index_read ----->
5'- AATGATACGGCGACCACCGAGATCTACAC -3'  5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'      5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                      3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'  
                                                                                          <----- nXTv2_read2
sequences to order:

T7-nXTv2_i5-1-top   5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
T7-nXTv2_i5-2-top   5'- CATGGAATTAATACGACTCACTATAGGGAGACTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
P-T7tspn-bot        5'- /5Phos/CTGTCTCTTATACACATCT -3'
P-sss_nXTv2_prmr    5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
nXTv2_i7_top        5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        
P-nXTv2_i7_bot      5'- /5Phos/CTGTCTCTTATACACATCT -3'
i5_PCR_ILAdptr      5'- AATGATACGGCGACCACCGA -3'
i7_PCR_ILAdptr      5'- CAAGCAGAAGACGGCATACGA -3'
i502_T7-1_shrt      5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC -3'
i503_T7-1_shrt      5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC -3'
i505_T7-1_shrt      5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC -3'
i506_T7-1_shrt      5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC -3'
i502_T7-1_long      5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGCGTC -3'
i503_T7-1_long      5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGCGTC -3'
i505_T7-1_long      5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGCGTC -3'
i506_T7-1_long      5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGCGTC -3'
i701_long           5'- CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG -3'
i702_long           5'- CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG -3'
i703_long           5'- CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG -3'
i704_long           5'- CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG -3'

sequencing primers used:
nXTv2_i5_index      5'- AATGATACGGCGACCACCGAGATCTACAC -3'
nXTv2_i7_index      5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
nXTv2_read1         5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
nXTv2_read2         5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'
Modified Nextera XT V2 i5 barcode sequences and adaptors

num.	i5_barcode	i5_name	i5_smplsheet	i5_full_sequence_(5'_->_3')
1	CTCTCTAT	S502_T7-1_shrt	CTCTCTAT	AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC
2	TATCCTCT	S503_T7-1_shrt	TATCCTCT	AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC
3	GTAAGGAG	S505_T7-1_shrt	GTAAGGAG	AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC
4	ACTGCATA	S506_T7-1_shrt	ACTGCATA	AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC
5	AAGGAGTA	S507_T7-1_shrt	AAGGAGTA	AATGATACGGCGACCACCGAGATCTACACAAGGAGTAGGGAGATCGTCGGCAGC
6	CTAAGCCT	S508_T7-1_shrt	CTAAGCCT	AATGATACGGCGACCACCGAGATCTACACCTAAGCCTGGGAGATCGTCGGCAGC
7	CGTCTAAT	S510_T7-1_shrt	CGTCTAAT	AATGATACGGCGACCACCGAGATCTACACCGTCTAATGGGAGATCGTCGGCAGC
8	TCTCTCCG	S511_T7-1_shrt	TCTCTCCG	AATGATACGGCGACCACCGAGATCTACACTCTCTCCGGGGAGATCGTCGGCAGC
9	TCGACTAG	S513_T7-1_shrt	TCGACTAG	AATGATACGGCGACCACCGAGATCTACACTCGACTAGGGGAGATCGTCGGCAGC
10	TTCTAGCT	S515_T7-1_shrt	TTCTAGCT	AATGATACGGCGACCACCGAGATCTACACTTCTAGCTGGGAGATCGTCGGCAGC
11	CCTAGAGT	S516_T7-1_shrt	CCTAGAGT	AATGATACGGCGACCACCGAGATCTACACCCTAGAGTGGGAGATCGTCGGCAGC
12	GCGTAAGA	S517_T7-1_shrt	GCGTAAGA	AATGATACGGCGACCACCGAGATCTACACGCGTAAGAGGGAGATCGTCGGCAGC
13	CTATTAAG	S518_T7-1_shrt	CTATTAAG	AATGATACGGCGACCACCGAGATCTACACCTATTAAGGGGAGATCGTCGGCAGC
14	AAGGCTAT	S520_T7-1_shrt	AAGGCTAT	AATGATACGGCGACCACCGAGATCTACACAAGGCTATGGGAGATCGTCGGCAGC
15	GAGCCTTA	S521_T7-1_shrt	GAGCCTTA	AATGATACGGCGACCACCGAGATCTACACGAGCCTTAGGGAGATCGTCGGCAGC
16	TTATGCGA	S522_T7-1_shrt	TTATGCGA	AATGATACGGCGACCACCGAGATCTACACTTATGCGAGGGAGATCGTCGGCAGC
original Nextera XT V2 i5/i7 barcode sequencers and primers

num.	i5_barcode	i5_name	i5_smplsheet	i5_full_sequence
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
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



UPDATE 8/27/2015

8/27/2015 T7-nXTv2_i5-1 testing

Validation of THS-seq update to Nextera XT V2 sequences[edit]

  • received tn5059 transposomes T7-nXTv2_i5-1 and nXTv2_i7 from Trina, will run through protocol and MiSeq testing for validation


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. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1
2. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2
3. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
4. i5XX-i7XX, T7-nXTv2_i5-1, 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 20 minutes at RT
a. Add 0.5 uL of 100% sterile glycerol to tube
b. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well
c. Add 1.0 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, T7-nXTv2_i5-1)
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 with Qubit or on TBU gel.



AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO

sample list
21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1
22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2
23. i503_shrt-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
24. i503_shrt-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
25. i505_shrt-i701_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
26. i505_shrt-i702_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
27. i506_long-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
28. i506_long-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
29. i502_long-i703_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
30. i502_long-i704_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
31. i503_shrt-i703_long, 8/27/2015, 4. T7-nXTv2_i5-1, 5X tn5-059, NTC
  • Amounts of RNA and water to add for MMLV RT (50 ng RNA)


9. single strand synthesis MMLV RT (Clontech)

20 uL reaction

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 fo 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 P-sss_nXTv2_prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since P-sss_nXTv2_prmr has a high Tm.

a. Add 2.5 uL of 20 uM P-sss_nXTv2_prmr 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 concentreate.

  • 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 vaccum 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 i5 AND i7 SEQUENCES!!!!!

Samples:
samples processed during RNA processing for rest ofjohn chang T cell samples, first cell division, day7 Tsle and Tmp, day 90 memory, AND nXTv2 THS-seq update RNA processing
21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1
22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2
23. i503_shrt-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
24. i503_shrt-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
25. i505_shrt-i701_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
26. i505_shrt-i702_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
27. i506_long-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
28. i506_long-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
29. i502_long-i703_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
30. i502_long-i704_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
31. i503_shrt-i703_long, 8/27/2015, 4. T7-nXTv2_i5-1, 5X tn5-059, NTC
KAPA SYBR FAST qPCR mix until saturation, X35 cycles

9  uL Taq2X
2  uL nXTv2_i5 index primer, 2 uL F, (i5XX_T7-1_shrt, OR long) 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, 59C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever
55C for comp ends for taq2X
HIGH BACKGROUND WITH 59C, NEXT TIME RUN AT 63C

  • 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.
' 5406.090161 5 '
3730.570111 2.5 in 10 uL
1. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 17614.20052 23.21540477 232.1540477
2. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 16680.7305 21.82259836 218.2259836
3. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 142250.0542 209.1813288 2091.813288
4. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, NTC 708.0800211 -2.009779054 -20.09779054


  • TBU gel after IVT, Guanidine HCl to remove proteins
  • yields from 500 cells approx. half that seen before, will try PAGE purified transposon instead, and v2 version.

File:ZhangLab 2 2015-08-28 11hr 27min-labeled.jpg


  • qPCR curves
  • do not gain much info from efficiency based on PCR curves for short versus long primers. seems 502 works better.

File:2015-09-09 nXTv2 validation, primers testing.bmp



  • after adding barcodes, TBE gel
  • high amount of primer dimers, maybe since ran at 59C for annealing instead of 63C. next time will try 63C.

File:ZhangLab 2 2015-09-09 18hr 14min-labeled.jpg

  • gel size selection amounts
' 6007.810179 2.5 for 150 ng X2
21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 23864.16071 23.11343865 6.48973103 12.97946206
22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 20633.37061 19.38380233 7.738419813 15.47683963
  • gel size selection

File:ZhangLab 2 2015-09-10 14hr 03min-labeled.jpg


  • gel size selection validation

File:ZhangLab 2 2015-09-10 18hr 23min-labeled.jpg

conclusions[edit]

  • submitted for sequencing


  • read stats:
BS-THS-nXTv2-valdation-i502-i701-Sep08_S35_L001_R1_00_trim.fastq
Total Reads: 2668419
Mapped Reads: 2443127
Clonal Reads: 587086
Total Unique Reads: 1856041

91.7% mapping rate
27.1% clonals rate

  • libraries nXTv2 transposome validated.
  • thus will use from now on for accessibility assay.

File:NXTv2-validation.png