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

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>Bsos
>Bsos
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*procotols from shendure paper, [http://genome.cshlp.org/content/early/2012/03/30/gr.136242.111?top=1 shendure paper transposition]
*procotols from shendure paper, [http://genome.cshlp.org/content/early/2012/03/30/gr.136242.111?top=1 shendure paper transposition]


*annealing of ME sequence to T7 transposon sequence
*annealing of ME sequence to T7 transposon sequence
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**2. 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.
**2. 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.
**3. cool to RT at 0.1 C/s
**3. cool to RT at 0.1 C/s


*transposome complex generation
*transposome complex generation
**1. incubate the below solutions at RT for 20 minutes
**1. add the below components into one tube and incubate for 20 minutes at RT
  2.5 uL of annealed transposon
  2.5 uL of annealed transposon
  2.5 uL of 100% sterile glycerol
  2.5 uL of 100% sterile glycerol
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(Tn5mC-A1top: 5’- GAT [5mC] TA [5mC] A[5mC] G [5mC] [5mC] T [5mC] [5mC] [5mC]
*transposition reaction
Downloaded from genome.cshlp.org on May 4, 2012 - Published by Cold Spring Harbor Laboratory Press
**add the below into one tube and incubate for 8 minutes at 55C.
T [5mC] G [5mC] G [5mC] [5mC] AT [5mC] AGA GAT GTG TAT AAG AGA CAG -3’,
1 uL nextera HMW buffer
IDT, annealed to Tn5mC-A1bot: 5’-[Phos]- CTG TCT CTT ATA CAC A -3’, IDT, by
1 uL genomic DNA at prepared quantities (1 ng)
incubating 10μl of each oligo at 100μM and 80μl of EB (QIAGen) at 95°C for 2 minutes
2 uL Nuclease free water
then cooling to RT at 0.1°C/s) with 2.5μl 100% glycerol and 5μl Ez-Tn5 transposase
1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST)
(Epicentre – Illumina) for 20 minutes at RT.
___________
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
 
 
*qiaquick AMPURE beads to clean up and get ready for IVT?
 
 


  should now be ready for IVT.
  should now be ready for IVT.
   
   
  ask rui
  ask rui



Revision as of 22:00, 7 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 NNNNNN
---NNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’               3'- CTACACATATTCTCTGTC ------NNNN


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

5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNN
3'- T[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC ------NNNN


NNNNN------ CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[CCCTATAGTGAGTCGTATTAA]T -3'
---NNNNNNNN 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


  • annealing of ME sequence to T7 transposon sequence
    • 1. Make 100 uM stock solution of T7tspn-top and T7tspn-bot.
    • 2. 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.
    • 3. cool to RT at 0.1 C/s


  • transposome complex generation
    • 1. 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


  • 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


  • qiaquick AMPURE beads to clean up and get ready for IVT?


should now be ready for IVT.


ask rui

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]