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

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>Bsos
>Bsos
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*would result in 100% efficiency of capture of sequence upon insertion, including single insertions.
*would result in 100% efficiency of capture of sequence upon insertion, including single insertions.


Summary:
Summary for using T7tspn-top:


  Sequences:
  Sequences:
   
   
        5’- [A][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top)  
5’- [A][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top)  
          spcr      T7 forward      spcr  read sequence  spcr        ME
    spcr      T7 forward      spcr  read sequence  spcr        ME  
5'- [ACATGAGA][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top2)
        spcr          T7 forward      spcr  read sequence  spcr        ME  
   
   
  5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot)
  5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot)
Line 53: Line 50:
   
   
  After IVT. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). All RNA strands will have same 5' end.
  After IVT. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). 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’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAA -3'
  RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN -3'
RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
   
   
   
   
  Adapters to add for first/second strand cDNA synthesis.
  Adapters to add for first/second strand cDNA synthesis.
   
   
          (second strand synthesis)
        (second strand synthesis)(T7tspn-sss)
       5'- A[TTAATACGACTCACTATAGGG]TCCT -3'  (T7tspn-sss)
       5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'   
  RNA  5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
  RNA  5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
                                                                                3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'  (T7tspn-fss)
                                                          3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'  (T7tspn-fss)
                                                                                      (first stand synthesis)
                                                                  (first stand synthesis)
   
   
  addition of lawn adapters through PCR
  addition of lawn adapters through PCR
   
   
                                                                                                                                        (T7tspn-law2)(illuminia bPCR adapter)
                                                                                                                    (T7tspn-law2)(illuminia bPCR adapter)
                                                                                                              3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                        3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                     5'- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAA -3'
                                     5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAA -3'
                                     3’- T[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTT -5'
                                     3’- CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTT -5'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]A[TTAATACGACTCACTATAGGG]TCCTCCCT -3'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'
      (illuminia bPCR adapter) (T7tspn-law1)          
    (illuminia bPCR adapter)(T7tspn-law1)    
                      
                      
   
   
Line 79: Line 80:
   read primers/amplification etc (only need 1 since only 1 5' end sequence)
   read primers/amplification etc (only need 1 since only 1 5' end sequence)
   
   
                                                                                                                        (T7tspn-Read2)
                                                                                                          (T7tspn-Read2)
         (illuminia bPCR adapter)                                                                             3'- TTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
         (illuminia bPCR adapter)                                                           3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3’
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3’
     [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]TAATTATGCTGAGTGATATCCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
     [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                          5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'                                            (illuminia bPCR adapter)
                                      5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'                                            (illuminia bPCR adapter)
                                                                        (T7tspn-Read1)   
                                                        (T7tspn-Read1)   
                                                                                                (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3'
                                                                              (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3'
                                                                                                                            (T7tspn-indx)
                                                                                                          (T7tspn-indx)
   
   
   
   
Summary for using with T7tspn-top2:
Sequences:
5'- [ACATGAGA][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top2)
        spcr          T7 forward      spcr  read sequence  spcr        ME
5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot)
After Annealing:
        5’- ACATGAGATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’        (T7tspn-top2)
                                                        3'- TCTACACATATTCTCTGTC [Phos]-5'  (T7tspn-bot)
5'- [phos] CTGTCTCTTATACACATCT -3'
      3’-  GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTAGAGTACA -5’
Fragments created after transposition:
5’- ACATGAGA[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATC -3'
                                                    3'- CTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]AGAGTACA -5’
Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:
NNNNN------ CTGTCTCTTATACACATC -3'              5’- ACATGAGA[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]AGAGTACA -5’              3'- CTACACATATTCTCTGTC ------NNNN
After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)
5’- ACATGAGA[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN
3'- TGTACTCT[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN
NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[CCCTATAGTGAGTCGTATTAA]TCTCATGT -3'
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]AGAGTACA -5’
After IVT. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). 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'
Adapters to add for first/second strand cDNA synthesis.
        (second strand synthesis)(T7tspn-sss)
      5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3' 
RNA  5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
                                                          3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'  (T7tspn-fss)
                                                                  (first stand synthesis)
addition of lawn adapters through PCR
                                                                                                                    (T7tspn-law2)(illuminia bPCR adapter)
                                                                                        3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                    5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAA -3'
                                    3’- CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTT -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'
    (illuminia bPCR adapter)(T7tspn-law1)   
                   
  read primers/amplification etc (only need 1 since only 1 5' end sequence)
                                                                                                          (T7tspn-Read2)
        (illuminia bPCR adapter)                                                          3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3’
    [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                      5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'                                            (illuminia bPCR adapter)
                                                        (T7tspn-Read1) 
                                                                              (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3'
                                                                                                          (T7tspn-indx)
===Experiments needed to be done for proof of concept/procedures===
===Experiments needed to be done for proof of concept/procedures===
   
   
Line 154: Line 245:
10. size selection.
10. size selection.


===ordered===
===ordered/to order===
  (T7tspn-top)  5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
  (T7tspn-top)  5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
  (T7tspn-bot)  5'- /5phos/CTGTCTCTTATACACATCT -3'
  (T7tspn-bot)  5'- /5phos/CTGTCTCTTATACACATCT -3'
  (T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (T7tspn-Read1)
  (T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (T7tspn-Read1)
   
   
  Ordered 2012-05-11 (need 5+ NT upstream of T7 for max efficiency)
  Need 5+ NT upstream of T7 for max efficiency
  (T7tspn-top2)  5'- ACATGAGATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
  (T7tspn-top2)  5'- ACATGAGATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
  (T7tspn-sss)  5'- A[TTAATACGACTCACTATAGGG]TCCT -3'
 
  (T7tspn-fss)  3'- V TTTTTTTTTTTTTTTTTTTT -5'  
For using with (T7-tspn-top) AND (T7-tspn-top2)
  (T7tspn-law1)  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]A[TTAATACGACTCACTATAGGG]TCCTCCCT -3'
  (T7tspn-sss)  5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'  
  (T7tspn-fss)  3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'
  (T7tspn-law1)  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'
  (T7tspn-law2)  3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
  (T7tspn-law2)  3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
  (T7tspn-Read2) 3'- TTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
  (T7tspn-Read2) 3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
  (T7tspn-indx)  5'- TTTTTTTTTTTTTTTTTCTGGC -3'
  (T7tspn-indx)  5'- TTTTTTTTTTTTTTTTTCTGGC -3'
 
 





Revision as of 00:27, 12 May 2012

Adaptation of transposition method for inserting T7

  • 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:

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)



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. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). 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'


Adapters to add for first/second strand cDNA synthesis.

        (second strand synthesis)(T7tspn-sss)
     5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'  
RNA  5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
                                                          3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'  (T7tspn-fss)
                                                                 (first stand synthesis)

addition of lawn adapters through PCR

                                                                                                                    (T7tspn-law2)(illuminia bPCR adapter)
                                                                                        3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                   5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAA -3'
                                   3’- CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTT -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'
    (illuminia bPCR adapter)(T7tspn-law1)     
                    



  read primers/amplification etc (only need 1 since only 1 5' end sequence)

                                                                                                          (T7tspn-Read2)
       (illuminia bPCR adapter)                                                           3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3’
    [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                      5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'                                            (illuminia bPCR adapter)
                                                       (T7tspn-Read1)  
                                                                              (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3'
                                                                                                          (T7tspn-indx)




Summary for using with T7tspn-top2:

Sequences:

5'- [ACATGAGA][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top2) 
        spcr          T7 forward      spcr   read sequence   spcr        ME 

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



After Annealing:

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

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


Fragments created after transposition:


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


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

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


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

5’- ACATGAGA[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN
3'- TGTACTCT[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN


NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[CCCTATAGTGAGTCGTATTAA]TCTCATGT -3'
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]AGAGTACA -5’


After IVT. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). 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'


Adapters to add for first/second strand cDNA synthesis.

        (second strand synthesis)(T7tspn-sss)
     5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'  
RNA  5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
                                                          3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'  (T7tspn-fss)
                                                                 (first stand synthesis)

addition of lawn adapters through PCR

                                                                                                                    (T7tspn-law2)(illuminia bPCR adapter)
                                                                                        3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                   5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAA -3'
                                   3’- CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTT -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'
    (illuminia bPCR adapter)(T7tspn-law1)     
                    



  read primers/amplification etc (only need 1 since only 1 5' end sequence)

                                                                                                          (T7tspn-Read2)
       (illuminia bPCR adapter)                                                           3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3’
    [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                      5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'                                            (illuminia bPCR adapter)
                                                       (T7tspn-Read1)  
                                                                              (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3'
                                                                                                          (T7tspn-indx)



Experiments needed to be done for proof of concept/procedures


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 adapters to 3' ends and PCR amplification


10. size selection.

ordered/to order

(T7tspn-top)   5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
(T7tspn-bot)   5'- /5phos/CTGTCTCTTATACACATCT -3'
(T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (T7tspn-Read1)

Need 5+ NT upstream of T7 for max efficiency
(T7tspn-top2)  5'- ACATGAGATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
For using with (T7-tspn-top) AND (T7-tspn-top2)
(T7tspn-sss)   5'- GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3' 
(T7tspn-fss)   3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'  
(T7tspn-law1)  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTA -3'
(T7tspn-law2)  3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
(T7tspn-Read2) 3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
(T7tspn-indx)  5'- TTTTTTTTTTTTTTTTTCTGGC -3'


left over notes

 Second adapter 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]