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

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  After fill in with T4 DNA polymerase, or BST or w/e
  After fill in with T4 DNA polymerase, or BST or w/e
   
   
  5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
  5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN
  3'- T[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC ------NNNN
  3'- T[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN
   
   
   
   
  NNNNN------ CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[CCCTATAGTGAGTCGTATTAA]T -3'
  NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[CCCTATAGTGAGTCGTATTAA]T -3'
  NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]A -5’
  NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]A -5’
   
   
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7. RNA fragmentation
7. RNA fragmentation
*Magnesium fragmentation or Shearing, look at what is done in RNA fragmentation kits





Revision as of 17:23, 8 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’- 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’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’               3'- CTACACATATTCTCTGTC ------NNNN


After fill in with T4 DNA polymerase, or BST or w/e

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


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


  read primers/amplification etc

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

  • 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


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.
  • 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.


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]