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:
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
- procotols from shendure paper, shendure paper transposition
- 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]