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

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Summary:
Summary:


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'- [CTACATGAGA][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top2)
        spcr          T7 forward      spcr  read sequence  spcr        ME
    
    
         5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
         5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’
Line 42: Line 44:
   
   
   
   
  After IVT. Addition of PolyA with E. coli PolyA polymerase (Ambion kit)
  After IVT. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). All RNA strands will have same 5' end.
   
   
  5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAA -3'
  RNA 5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAA -3'
   
   
   
   
  Adapters to add for PCR amplification.
  Adapters to add for first/second strand cDNA synthesis.
   
   
  5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAA -'3
          (second strand synthesis)
                                                                          5'- V TTTTTTTTTTTTTTTTTTTTTT[lawn thingy]
      5'- A[TTAATACGACTCACTATAGGG]TCCT -3'
RNA 5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAA -'3
                                                                                5'- V TTTTTTTTTTTTTTTTTTTTTT -3'
                                                                                      (first stand synthesis)
   
   
   read primers/amplification etc
addition of lawn adapters through PCR
                                                                                                                                                (illuminia bPCR adapter)
                                                                                                              3'- V TTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                    5'- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAA -3'
                                    3’- T[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTT -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]A[TTAATACGACTCACTATAGGG]TCCTCCCT -3'
      (illuminia bPCR adapter)                                                                   
   read primers/amplification etc (only need 1 since only 1 5' end sequence)
   
   
  5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’
  5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’

Revision as of 21:32, 11 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:

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

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

RNA 5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAA -3'


Adapters to add for first/second strand cDNA synthesis.

          (second strand synthesis)
     5'- A[TTAATACGACTCACTATAGGG]TCCT -3'
RNA  5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAA -'3
                                                                               5'- V TTTTTTTTTTTTTTTTTTTTTT -3'
                                                                                     (first stand synthesis)

addition of lawn adapters through PCR

                                                                                                                                                (illuminia bPCR adapter)
                                                                                                             3'- V TTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                   5'- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAA -3'
                                   3’- T[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTT -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]A[TTAATACGACTCACTATAGGG]TCCTCCCT -3'
      (illuminia bPCR adapter)                                                                     



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

5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’
                      5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (T7tspn-Read1)
                                                                           

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

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

Ordered 2012-05-11 (need 5+ NT upstream of T7 for max efficiency)
(T7tspn-top2)  5'- CTACATGAGATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -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]