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'- [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
- procotols from shendure paper, shendure paper transposition
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]