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

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>Bsos
>Bsos
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===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 33: Line 33:
   
   
  '''NEXTERA XT V2 R1 TOP (64 bp)'''
  '''NEXTERA XT V2 R1 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.  \-----------------/
Line 39: Line 39:
                                                                                    
                                                                                    
  '''NEXTERA XT V2 R1 TOP + EXTRA (69 bp)'''
  '''NEXTERA XT V2 R1 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.  \-----------------/
Line 47: Line 47:
  5'- [phos]CTGTCTCTTATACACATCT -3' (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 54:
   
   
   
   
  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 [Phos]-5' (T7tspn-bot)
   
   
Line 64: Line 64:
   
   
   
   
  Fragments created after transposition:
  '''Fragments created after transposition:'''
   
   
  5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNN--------- 5P/CTGTCTCTTATACACATCT -3'
  5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNN--------- 5P/CTGTCTCTTATACACATCT -3'
Line 70: Line 70:
   
   
   
   
  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 76:
   
   
   
   
  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 NNNNNNNNNNNNN -3'
Line 86: Line 86:
   
   
   
   
  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 95:
   
   
   
   
  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 101:
   
   
   
   
  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) (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' (Tm=70, anneal@64C) (sss_nXTv2_prmr)
  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' (nXTv2_i7_bot)
   
   
    5'- /5Phos/CTGTCTCTTATACACATCT -3'                (nXTv2_i7_bot)
          3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top)
             
   
   
    5'- /5Phos/CTGTCTCTTATACACATCT -3'               (nxtv2_i7_bot)
'''After custom nextera tagmentation:'''
          3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nxtv2_i7_top)
   
   
DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3'
cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
   
   
After custom nextera tagmentation:
   
   
DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3'
cDNA  3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
   
   
'''After fill in:'''
   
   
DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
   
   
After fill in:
   
   
  DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
  '''Amplification (to add i5/i7 barcodes):'''
  cDNA  3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
'''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 -3'                                  (i5_PCR_ILAdptr, Tm=59C)
  5’- [AATGATACGGCGACCACCGA]GGG]AGATCCTCCCTCGCGCCATCAGAG    T7-top2-PCR-iaf3 (comp part (Tm=77), all (Tm=85C)
  5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGC -3'    (i5_shrt, comp region Tm=60C, whole=76C)
                 
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5_long, comp region Tm=65C, whole=77C)
    5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAG    T7-top2-PCR-iaf2 (comp part (Tm=73), all (Tm=83C))
      5’- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5_orig, comp region Tm=56C, whole=75C)
                          X X
                                      XXXXXX
    5’- [AATGATACGGCGACCACCGA]GATCTCTCCCTCGCGCCATCAGAGAT  T7-top2-PCR-iaf (original) (comp region 70C) (whole Tm=83C)  
                      DNA 5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                          X X XXXXX
                      cDNA       3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                DNA /5Phos/GGG]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                                                              (i7_orig, comp region Tm=57C, whole=73C)  3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                cDNA   3'- CCC]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                                                              (i7_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (custom NXTA barcodes) (Tm=86C)
                                                              (i5_PCR_ILAdptr, Tm=58C)                                         3'- AGCATACGGCAGAAGACGAAC -5'
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                                                        3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)
   
   
  Read Primers:
  '''Read Primers:'''
'''Using i5_shrt or i5_long and i7_long'''
    
    
Using T7-top2-PCR-iaf2 for amp.
   
   
                                          (Nextera_R1)
          nXT i5 index read ----->                          nXT read1 ----->                        nXT i7 index read ----->
                          5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
5'- AATGATACGGCGACCACCGAGATCTACAC -3' 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'      5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                                        (T7tspn-Read1)                                  (Nextera_Ind index read primer)
  5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
                          5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'      5'- CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG -3'
  3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
  5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG[XXXXXX]ATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                      3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
  3'- [TTACTATGCCGCTGGTGGCT]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                          <----- read2 nXT

Revision as of 00:43, 5 August 2015

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



  • 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.


  • Thus will redesign THS-seq using Nextera XT V2 sequencing/index/flowcell/amplification primers.


Sequences information, Using T7-nXTv2_i5-1-top and T7-nXTv2_i5-2-top

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 

NEXTERA XT V2 R1 TOP (64 bp)
5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’            (T7-nXTv2_i5-1-top)
     \---/  \-/\---------------------/ \--------------------------------/
     spcr T7 rec.  T7 req. forward         read seq.  \-----------------/
                                                              ME  
                                                                                 
NEXTERA XT V2 R1 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  


5'- [phos]CTGTCTCTTATACACATCT -3' (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 [Phos]-5' (T7tspn-bot)


5'- [phos] 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 NNNNNNNNNNNNN -3'
3'- GTACC[TTAATTATGCTGAGTGATATCCCTCT]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNN)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) (sss_nXTv2_prmr)
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


Now have double stranded DNA, which should be long.

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' (Tm=70, anneal@64C) (sss_nXTv2_prmr)
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
Custom Nextera i7 transposome
   i7 adapter
       
   5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        (nXTv2_i7_top)
                  3'- TCTACACATATTCTCTGTC/5Phos/ -5' (nXTv2_i7_bot)


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

After custom nextera tagmentation:

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN 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'    (i5_shrt, comp region Tm=60C, whole=76C)
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5_long, comp region Tm=65C, whole=77C)
      5’- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5_orig, comp region Tm=56C, whole=75C)
                                     XXXXXX
                     DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                     cDNA        3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                              (i7_orig, comp region Tm=57C, whole=73C)   3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                              (i7_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                              (i5_PCR_ILAdptr, Tm=58C)                                         3'- AGCATACGGCAGAAGACGAAC -5'

Read Primers:
Using i5_shrt or i5_long and i7_long
 

         nXT i5 index read ----->                           nXT read1 ----->                        nXT 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'
                                                                                          <----- read2 nXT