Brandon:LabNotes/Project1/2012-5-7: Difference between revisions

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Line 58: Line 58:
   
   
   
   
  RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN -3'
  RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
   
   
  RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
  RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
Line 65: Line 65:
  Adaptors to add for first/second strand cDNA synthesis.
  Adaptors to add for first/second strand cDNA synthesis.
   
   
    (primer 1, from nextera)
  5'- AATGATACGGCGACCACCGA -3' (second strand synthesis)(T7tspn-sss)
POLY-A METHOD
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3' 
Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors
                              RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' (T7tspn-fss)
                                                                                        3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
      (second strand synthesis)  
                                                                                              (first stand synthesis)           3'- AGCATACGGCAGAAGACGAAC -5'
        ILA adaptor blue      (T7-top2-PCR-iaf)Tm=86.59
                                                                                                                                    (primer 2, from nextera)
  5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=66.79 comp region only)
                            XXXX
                    RNA 5’- GGGTC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAA -3' (polyA only Tm=49-52)
                                                      (T20VN_PE_R)(Tm=78.92)  3'- NV TTTTTTTTTTTTTTTTTTTTTCTAGCC[TTCTCGCCAAGTCGTCCTTACG]-Bio-5'
                                                                                PCR_R.N2Ind[XX] (Tm=86.32) 3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                                    adaptor2 Tm=64.41    spr    bc      ILA adaptor orange
   
   
   
   
RANDOM NONAMER METHOD
  Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors
   
   
  read primers/amplification etc (only need 1 since only 1 5' end sequence)
      (second strand synthesis)
        ILA adaptor blue      (T7-top2-PCR-iaf)Tm=86.59
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT]  -3’(Tm=66.79 comp region only)
                            XXXX
                    RNA 5’- GGGTC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN... -3'
                                                                                                  XXXXXX XXXXXXXXXXXX
                                                                    (N9_PE_R)(Tm=low) 3'-NNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG] -5'-Bio
                                                                        PCR_R.N2Ind[XX] (Tm=86.32) 3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                            adaptor2 Tm=64.41    spr    bc      ILA adaptor orange
   
   
                                                                                                          (T7tspn-Read2)
        (illuminia bPCR adaptor)                                                          3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3’
    [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                      5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'                                            (illuminia bPCR adaptor)
                                                        (T7tspn-Read1) 
                                                                              (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3'
                                                                                                          (T7tspn-indx)
   
   
   
   
 
 
===Summary for using with T7tspn-top2: (USED)===
===Summary for using with T7tspn-top2:===
   
   
  Sequences:
  Sequences:
Line 117: Line 124:
   
   
   
   
  5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATC -3'
  5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3'
                                                      3'- CTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’  
                                                    3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’  
   
   
   
   
  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:
   
   
  NNNNN------ CTGTCTCTTATACACATC -3'               5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
  NNNNN------ CTGTCTCTTATACACATCT -3'             5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
  NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’               3'- CTACACATATTCTCTGTC ------NNNN
  NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’             3'- TCTACACATATTCTCTGTC ------NNNN
   
   
   
   
Line 143: Line 150:
  All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7.
  All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7.
   
   
  RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN -3'
  RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
   
   
  RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
  RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
T7-frag-block-top2
CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/
POLY-A METHOD
Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors
      (second strand synthesis)
        ILA adaptor blue      (T7-top2-PCR-iaf)Tm=86.59
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT]  -3’(Tm=66.79 comp region only)
                      X X  XXXXX
              RNA 5’- GGG][AGATC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAA -3'  (polyA only Tm=49-52)
                                                      (T20VN_PE_R)(Tm=78.92)  3'- NV TTTTTTTTTTTTTTTTTTTTTCTAGCC[TTCTCGCCAAGTCGTCCTTACG]-Bio-5'
                                                                                PCR_R.N2Ind[XX] (Tm=86.32)  3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][XXXXXX][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                                    adaptor2 Tm=64.41    spr    bc      ILA adaptor orange
Sequencing primers:
T7tspn-read1 was designed off of template by mistake. however did not make a difference.
          (Nextera_R1)  5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
                            XX
        (T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'                                          (N2IndSeq)
                            XX                                                                        5'- AAGAGCGGTTCAGCAGGAATGCCGAG -3'
5’- AATGATACGGCGACCACCGAGATCTCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGAGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                         
RANDOM NONAMER METHOD
  Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors
      (second strand synthesis)
        ILA adaptor blue      (T7-top2-PCR-iaf)Tm=86.59
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’(Tm=66.79 comp region only)
                      X X  XXXXX
              RNA 5’- GGG][AGATC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN... -3'
                                                                                                  XXXXXX XXXXXXXXXXXX
                                                                    (N9_PE_R)(Tm=low) 3'-NNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG] -5'-Bio
                                                                        PCR_R.N2Ind[XX] (Tm=86.32)  3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                            adaptor2 Tm=64.41    spr    bc      ILA adaptor orange
  RANDOM NONAMER METHOD
  Sequencing adaptors
      (second strand synthesis)          T7tspn-Read1 (Tm=75.80)
        ILA adaptor blue      TC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG 
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNN GATCGG[AAGAGCGGTTCAGCAGGAATGC][CGAG][CTTGTA][ATCTCGTATGCCGTCTTCTGCTTG] -5'
    [TTACTATGCCGCTGGTGGCT][CTAGA][GAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC]
                                                                                                            adaptor2 Tm=64.41    spr    bc      ILA adaptor orange
read primers from sequence reads etc
===Summary for using with T7tspn-top3:===
Sequences:
5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][CATTA][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top3)
        spcr      T7 forward  [req spcr] [rd spcr]      ME
5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot)
G][GGAGA][CATTA][AGATGTGTATAAGAGACAG  (Tm 60.71)
  T7 description:
                                                        +1
                              CATGAGA[TTAATACGACTCACTATA|G|GGAGA]
[end sequence for optimal binding]        T7 -->          [front sequence for optimal binding]
After Annealing:
                      5'- CATGAGATTAATACGACTCACTATAGGGAGACATTAAGATGTGTATAAGAGACAG -3’        (T7tspn-top3)
                                                          3'- TCTACACATATTCTCTGTC [Phos]-5'  (T7tspn-bot)
5'- [phos] CTGTCTCTTATACACATCT -3'
        3’- GACAGAGAATATGTGTAGAATTACAGAGGGATATCACTCAGCATAATTAGAGTAC -5’
Fragments created after transposition:
5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3'
                                      3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’
Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:
NNNNN------ CTGTCTCTTATACACATCT -3'              5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNNNNNN
NNNNNNNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’              3'- TCTACACATATTCTCTGTC ------NNNN
After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)
5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNNNNNN
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]GTAATTCTACACATATTCTCTGTC (NNNNNN)NNNN
NNN(NNNNNN) CTGTCTCTTATACACATCTTAATG[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
NNNNNNNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’
After IVT, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit).
Can also try adding adapter to 3'OH end with T4 RNA ligase.
All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7.
RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN -3'
RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
   
   
   
   
Line 151: Line 276:
  Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors
  Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors
   
   
  (primer 1, from nextera)
5'- AATGATACGGCGACCACCGA -3' (second strand synthesis)(T7tspn-sss)
5'-[AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTA -3' 
                              RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'      (illuminia bPCR adaptor)
                                                                            (T7tspn-fss)  3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                        (first stand synthesis)                                  3'- AGCATACGGCAGAAGACGAAC -5'
                                                                                                                                    (primer 2, from nextera)
                                                                                                           
   
   
 
POLY-A METHOD
Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors
   
   
  read primers/amplification etc (only need 1 since only 1 5' end sequence)
        (second strand synthesis)
                                      (Tm=60.71 comp region only)
    ILA adaptor blue  (T7-top3-PCR-iaf) Tm=81.40
5'- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3'
                          RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN AAAAAAAAAAAAAAAAAAAAA -3' (polyA only Tm=49-52)
                                              (T20VN_PE_R)(Tm=78.92)  3'- NV TTTTTTTTTTTTTTTTTTTTTCTAGCC[TTCTCGCCAAGTCGTCCTTACG]-Bio-5'
                                                                        PCR_R.N2Ind[XX] (Tm=86.32) 3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                          adaptor2 Tm=64.41      spr    bc      ILA adaptor orange
   
   
                                                                                                          (T7tspn-Read2)
        (illuminia bPCR adaptor)                                                            3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3’
    [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                      5'- AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'                                            (illuminia bPCR adaptor)
                                                        (T7tspn-Read1-2) 
                                                                                (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3'
                                                                                                              (T7tspn-indx)
   
   
RANDOM NONAMER METHOD
  Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors
        (second strand synthesis)
                                      (Tm=60.71 comp region only)
    ILA adaptor blue  (T7-top3-PCR-iaf) Tm=81.40
5'- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3'
                          RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -3'
                                                                                          XXXXXX XXXXXXXXXXXX
                                                          (N9_PE_R)(Tm=low) 3'-NNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG] -5'-Bio
                                                                PCR_R.N2Ind[XX] (Tm=86.32)  3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                    adaptor2 Tm=64.41    spr    bc      ILA adaptor orange
 
 
   
   


===Experiments needed to be done for proof of concept/procedures===
===Experiments needed to be done for proof of concept/procedures===
Line 243: Line 377:
  (T7tspn-top)  5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
  (T7tspn-top)  5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
  (T7tspn-bot)  5'- /5phos/CTGTCTCTTATACACATCT -3'
  (T7tspn-bot)  5'- /5phos/CTGTCTCTTATACACATCT -3'
For using with (T7tspn-top)
  (T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
  (T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
(T7-sss-1)    5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGA -3' 
   
   
  For using with(T7-top2)
  For using with(T7-top2)
Line 252: Line 391:
  (T7-fss)        3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
  (T7-fss)        3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
    
    
  (T7-fss-shrt)    3'- V TTTTTTTTTTTTTTTTTTTTTTTT
  (T7-fss-shrt)    3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'
    
    
  (T7-sss)         5'-[AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTA -3'  
  (T7-sss-2)       5'-[AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGA -3'  
    
    
  (T7-Nprimer1)    5'- AATGATACGGCGACCACCGA -3'
  (T7-Nprimer1)    5'- AATGATACGGCGACCACCGA -3'
Line 265: Line 404:
    
    
  (T7tspn-indx)    5'- TTTTTTTTTTTTTTTTTCTGGC -3'
  (T7tspn-indx)    5'- TTTTTTTTTTTTTTTTTCTGGC -3'
quick and dirty validation of IVT RNA fragments with RT-PCR, For using with(T7-top2) and (T7tspn-top).
(tspn-vldte-RT) (add on unique sequence to end as primer for PCR, since both ends are the same)
5'- [CAAGCAGAAGACGGCATACGAGAT]GATGTGTATAAGAGACAG -3'
      (illuminia bPCR adaptor)    ME
(tspn-vldate-R)
5'- CAAGCAGAAGACGGCATACGA -3' (primer 2, from nextera)
(tspn-vldte-F)
5'- TCCTCCCTCGCGCCATCAGAGAT -3'


===left over notes===
===left over notes===

Latest revision as of 23:49, 11 February 2014

Adaptation of transposition method for inserting T7[edit]

  • in reference to shendure paper transposition
  • also in reference to Athurva's summary
  • new technique of only annealing sequences for form full mosaic ends and adding adaptors.
  • would result in 100% efficiency of capture of sequence upon insertion, including single insertions.

Summary for using T7tspn-top:[edit]

Sequences:

5’- [A][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top) 
   spcr      T7 forward      spcr   read sequence   spcr        ME 

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

T7 description:
                                                        +1
                             CATGAGA[TTAATACGACTCACTATA|G|GGAGA]
[end sequence for optimal binding]         T7 -->          [front sequence for optimal binding]


After Annealing:

       5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
                                                3'- TCTACACATATTCTCTGTC [Phos]-5'   (T7tspn-bot)

5'- [phos] CTGTCTCTTATACACATCT -3'
      3’-  GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’


Fragments created after transposition:


5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATC -3'
                                            3'- CTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]A -5’ 


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

NNNNN------ CTGTCTCTTATACACATC -3'               5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]A -5’               3'- CTACACATATTCTCTGTC ------NNNN


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

5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN
3'- T[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN


NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[CCCTATAGTGAGTCGTATTAA]T -3'
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]A -5’


After IVT, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). 
Can also try adding adapter to 3'OH end with T4 RNA ligase.
All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7.


RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'

RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'


Adaptors to add for first/second strand cDNA synthesis.


POLY-A METHOD
Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors

      (second strand synthesis) 
       ILA adaptor blue      (T7-top2-PCR-iaf)Tm=86.59
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=66.79 comp region only)
                            XXXX
                   RNA 5’- GGGTC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAA -3' (polyA only Tm=49-52)
                                                     (T20VN_PE_R)(Tm=78.92)  3'- NV TTTTTTTTTTTTTTTTTTTTTCTAGCC[TTCTCGCCAAGTCGTCCTTACG]-Bio-5'
                                                                               PCR_R.N2Ind[XX] (Tm=86.32)  3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                                   adaptor2 Tm=64.41     spr    bc      ILA adaptor orange



RANDOM NONAMER METHOD 
 Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors 

      (second strand synthesis) 
       ILA adaptor blue      (T7-top2-PCR-iaf)Tm=86.59
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT]  -3’(Tm=66.79 comp region only)
                            XXXX
                   RNA 5’- GGGTC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN... -3'
                                                                                                  XXXXXX XXXXXXXXXXXX
                                                                   (N9_PE_R)(Tm=low) 3'-NNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG] -5'-Bio
                                                                        PCR_R.N2Ind[XX] (Tm=86.32)  3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                            adaptor2 Tm=64.41     spr    bc      ILA adaptor orange




Summary for using with T7tspn-top2: (USED)[edit]

Sequences:

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

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

 T7 description:
                                                        +1
                              CATGAGA[TTAATACGACTCACTATA|G|GGAGA]
[end sequence for optimal binding]         T7 -->          [front sequence for optimal binding]



After Annealing:

       5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’         (T7tspn-top2)
                                                         3'- TCTACACATATTCTCTGTC [Phos]-5'   (T7tspn-bot)

5'- [phos] CTGTCTCTTATACACATCT -3'
      3’-  GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTAGAGGGATATCACTCAGCATAATTAGAGTAC -5’


Fragments created after transposition:


5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3'
                                                    3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 


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

NNNNN------ CTGTCTCTTATACACATCT -3'              5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’              3'- TCTACACATATTCTCTGTC ------NNNN



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

5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN


NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’



After IVT, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). 
Can also try adding adapter to 3'OH end with T4 RNA ligase.
All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7.

RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'

RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'

T7-frag-block-top2 
CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/

POLY-A METHOD
Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors

      (second strand synthesis) 
       ILA adaptor blue      (T7-top2-PCR-iaf)Tm=86.59
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT]  -3’(Tm=66.79 comp region only)
                      X X  XXXXX
              RNA 5’- GGG][AGATC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAA -3'  (polyA only Tm=49-52)
                                                     (T20VN_PE_R)(Tm=78.92)  3'- NV TTTTTTTTTTTTTTTTTTTTTCTAGCC[TTCTCGCCAAGTCGTCCTTACG]-Bio-5'
                                                                               PCR_R.N2Ind[XX] (Tm=86.32)  3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][XXXXXX][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                                   adaptor2 Tm=64.41     spr    bc      ILA adaptor orange

Sequencing primers:
T7tspn-read1 was designed off of template by mistake. however did not make a difference.

         (Nextera_R1)  5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
                           XX
        (T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'                                           (N2IndSeq)
                           XX                                                                         5'- AAGAGCGGTTCAGCAGGAATGCCGAG -3'
5’- AATGATACGGCGACCACCGAGATCTCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGAGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                          


RANDOM NONAMER METHOD 
 Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors 

      (second strand synthesis) 
       ILA adaptor blue      (T7-top2-PCR-iaf)Tm=86.59
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’(Tm=66.79 comp region only)
                      X X  XXXXX
              RNA 5’- GGG][AGATC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN... -3'
                                                                                                  XXXXXX XXXXXXXXXXXX
                                                                   (N9_PE_R)(Tm=low) 3'-NNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG] -5'-Bio
                                                                        PCR_R.N2Ind[XX] (Tm=86.32)  3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                            adaptor2 Tm=64.41     spr    bc      ILA adaptor orange



 RANDOM NONAMER METHOD 
 Sequencing adaptors 

      (second strand synthesis)           T7tspn-Read1 (Tm=75.80) 
       ILA adaptor blue       TC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG   
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNN GATCGG[AAGAGCGGTTCAGCAGGAATGC][CGAG][CTTGTA][ATCTCGTATGCCGTCTTCTGCTTG] -5'
    [TTACTATGCCGCTGGTGGCT][CTAGA][GAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC]
                                                                                                            adaptor2 Tm=64.41     spr    bc      ILA adaptor orange





read primers from sequence reads etc

Summary for using with T7tspn-top3:[edit]

Sequences:

5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][CATTA][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top3)
       spcr      T7 forward   [req spcr] [rd spcr]       ME 

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


G][GGAGA][CATTA][AGATGTGTATAAGAGACAG  (Tm 60.71)

 T7 description:
                                                        +1
                              CATGAGA[TTAATACGACTCACTATA|G|GGAGA]
[end sequence for optimal binding]         T7 -->          [front sequence for optimal binding]



After Annealing:

                     5'- CATGAGATTAATACGACTCACTATAGGGAGACATTAAGATGTGTATAAGAGACAG -3’         (T7tspn-top3)
                                                         3'- TCTACACATATTCTCTGTC [Phos]-5'   (T7tspn-bot)

5'- [phos] CTGTCTCTTATACACATCT -3'
       3’- GACAGAGAATATGTGTAGAATTACAGAGGGATATCACTCAGCATAATTAGAGTAC -5’


Fragments created after transposition:


5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3'
                                      3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 


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

NNNNN------ CTGTCTCTTATACACATCT -3'               5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNNNNNN
NNNNNNNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’               3'- TCTACACATATTCTCTGTC ------NNNN



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

5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNNNNNN
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]GTAATTCTACACATATTCTCTGTC (NNNNNN)NNNN


NNN(NNNNNN) CTGTCTCTTATACACATCTTAATG[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
NNNNNNNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’



After IVT, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). 
Can also try adding adapter to 3'OH end with T4 RNA ligase.
All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7.

RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN -3'

RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'



Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors


 
POLY-A METHOD
Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors

       (second strand synthesis)
                                     (Tm=60.71 comp region only)
    ILA adaptor blue  (T7-top3-PCR-iaf) Tm=81.40
5'- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3'
                          RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN AAAAAAAAAAAAAAAAAAAAA -3' (polyA only Tm=49-52)
                                             (T20VN_PE_R)(Tm=78.92)  3'- NV TTTTTTTTTTTTTTTTTTTTTCTAGCC[TTCTCGCCAAGTCGTCCTTACG]-Bio-5'
                                                                       PCR_R.N2Ind[XX] (Tm=86.32)  3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                          adaptor2 Tm=64.41      spr    bc      ILA adaptor orange


RANDOM NONAMER METHOD
 Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors 

       (second strand synthesis)
                                     (Tm=60.71 comp region only)
    ILA adaptor blue  (T7-top3-PCR-iaf) Tm=81.40
5'- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3'
                          RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -3'
                                                                                         XXXXXX XXXXXXXXXXXX
                                                          (N9_PE_R)(Tm=low) 3'-NNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG] -5'-Bio
                                                               PCR_R.N2Ind[XX] (Tm=86.32)  3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                   adaptor2 Tm=64.41     spr    bc      ILA adaptor orange


 
 

Experiments needed to be done for proof of concept/procedures[edit]


1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of T7tspn-top and T7tspn-bot.
    • b. Incubate 10 uL of each oligo (100uM) with 80 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 100 uL.
    • c. cool to RT at 0.1 C/s


2. transposome complex generation, run controls!!! (nextera normal, without transposon, without transposase, Jurkat DNA only, transposon only)

  • add the below components into one tube and incubate for 20 minutes at RT
2.5 uL of annealed transposon
2.5 uL of 100% sterile glycerol
5.0 uL of Ez-TN5 transposase
  • store at -20, is good for a year

3. transposition reaction

  • add the below into one tube and incubate for 8 minutes at 55C.
1 uL nextera HMW buffer
1 uL genomic DNA at prepared quantities (1 ng)
2 uL Nuclease free water
1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST)
___________
5 uL total solution
method used in shendure paper:
4 uL nextera HMW buffer
X uL genomic DNA at prepared quantities
X uL Nuclease free water
______
17.5 uL total solution

add 2.5 uL of prepared transposomes


4. 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: 50 C 10 minutes, 70 C 20 minutes


5. Fill in reaction

  • Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
  • IVT on filled in reaction and not filled in reaction in to show fill-in is working.


6. should now be ready for IVT.

  • Look at Ambion's, life tech's and NEB's T7s. for which one to buy.


7. RNA fragmentation

  • Magnesium fragmentation or Shearing, look at what is done in RNA fragmentation kits


8. PolyA polymerase and ATP to A-tail 3'-ends


9. addition of adaptors to 3' ends and PCR amplification


10. size selection.

ordered/to order[edit]

(T7tspn-top)   5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
(T7tspn-bot)   5'- /5phos/CTGTCTCTTATACACATCT -3'

For using with (T7tspn-top)

(T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'

(T7-sss-1)     5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGA -3'  

For using with(T7-top2)

                 Need 5+ NT upstream of T7 for max efficiency
(T7-top2)        5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
 
(T7-fss)         3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
 
(T7-fss-shrt)    3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'
 
(T7-sss-2)       5'-[AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGA -3' 
 
(T7-Nprimer1)    5'- AATGATACGGCGACCACCGA -3'
 
(T7-Nprimer2)    3'- AGCATACGGCAGAAGACGAAC -5'
 
(T7tspn-Read1-2) 5'- AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
 
(T7tspn-Read2)   3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
 
(T7tspn-indx)    5'- TTTTTTTTTTTTTTTTTCTGGC -3'


quick and dirty validation of IVT RNA fragments with RT-PCR, For using with(T7-top2) and (T7tspn-top).

(tspn-vldte-RT) (add on unique sequence to end as primer for PCR, since both ends are the same)
5'- [CAAGCAGAAGACGGCATACGAGAT]GATGTGTATAAGAGACAG -3'
     (illuminia bPCR adaptor)     ME

(tspn-vldate-R)
5'- CAAGCAGAAGACGGCATACGA -3' (primer 2, from nextera)

(tspn-vldte-F)
5'- TCCTCCCTCGCGCCATCAGAGAT -3'

left over notes[edit]

 Second adaptor to anneal:

                                                                    [phos] -5' CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’
5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNN------ CTGTCTCTTATACACATC -3'    X   X XXX X XXXX X XX  XX
             XX  XX X XXXX X XXX X   X    3'- CTACACATATTCTCTGTC ------NNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’ 
         3'- CTAGCCAGACGGAACGGTCGGGCGAGGTAGTCTCTACACATATTCTCTGTC [phos] -5' 


Strands after fill-in, ligation, separation

top strand
5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’

bottom strand
3'- CTAGCCAGACGGAACGGTCGGGCGAGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’ 


to stop nonspecific amplification in shendure protocol 
5’-[Phos]- CTGTCTCTTATACACATC -3’ [ddC - 3' to block ends]