Brandon:LabNotes/Project1/2012-5-7

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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)

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. 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'


Adaptors 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 adaptors through PCR

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



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

                                                                                                          (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:

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------ CTGTCTCTTATACACATC -3'
                                                     3'- CTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 


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
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’               3'- CTACACATATTCTCTGTC ------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. 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’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN -3'

RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'


Adaptors to add for first/second strand cDNA synthesis.

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

addition of lawn adaptors through PCR

                                                                                                                     (T7tspn-law2)(illuminia bPCR adaptor)
                                                                                           3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                   5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
                                   3’- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTT -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTA -3'
    (illuminia bPCR adaptor)(T7tspn-law1)     
                    



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

                                                                                                          (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)  
                                                                                (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 adaptors 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'

Need 5+ NT upstream of T7 for max efficiency
(T7tspn-top2)  5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
For using with (T7-tspn-top) AND (T7-tspn-top2)
(T7tspn-sss)     5'- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTA -3'
(T7tspn-fss)     3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5'  
(T7tspn-law1)    5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTA -3'
(T7tspn-law2)    3'- V TTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
(T7tspn-Read2)   3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5'
(T7tspn-indx)    5'- TTTTTTTTTTTTTTTTTCTGGC -3'
(T7tspn-read1-2) 5'- AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'


left over notes

 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]