Brandon:LabNotes/Project1/2012-5-7: Difference between revisions
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(primer 1, from nextera) | (primer 1, from nextera) | ||
5'- AATGATACGGCGACCACCGA -3' (second strand synthesis)(T7tspn-sss) | 5'- AATGATACGGCGACCACCGA -3' (second strand synthesis)(T7tspn-sss) | ||
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT] | 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGA -3' | ||
RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' (T7tspn-fss) | RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' (T7tspn-fss) | ||
3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' | 3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' | ||
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(index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3' | (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3' | ||
(T7tspn-indx) | (T7tspn-indx) | ||
===Summary for using with T7tspn-top2:=== | ===Summary for using with T7tspn-top2:=== |
Revision as of 20:58, 16 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 for using T7tspn-top:
Sequences: 5’- [A][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top) spcr T7 forward spcr read sequence spcr ME 5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot) T7 description: +1 CATGAGA[TTAATACGACTCACTATA|G|GGAGA] [end sequence for optimal binding] T7 --> [front sequence for optimal binding] After Annealing: 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, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). Can also try adding adapter to 3'OH end with T4 RNA ligase. All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7. RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN -3' RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' Adaptors to add for first/second strand cDNA synthesis. (primer 1, from nextera) 5'- AATGATACGGCGACCACCGA -3' (second strand synthesis)(T7tspn-sss) 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGA -3' RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' (T7tspn-fss) 3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' (first stand synthesis) 3'- AGCATACGGCAGAAGACGAAC -5' (primer 2, from nextera) read primers/amplification etc (only need 1 since only 1 5' end sequence) (T7tspn-Read2) (illuminia bPCR adaptor) 3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5' 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3’ [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (illuminia bPCR adaptor) (T7tspn-Read1) (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3' (T7tspn-indx)
Summary for using with T7tspn-top2:
Sequences: 5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top2) spcr T7 forward required spcr read sequence spcr ME 5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot) T7 description: +1 CATGAGA[TTAATACGACTCACTATA|G|GGAGA] [end sequence for optimal binding] T7 --> [front sequence for optimal binding] After Annealing: 5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ (T7tspn-top2) 3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot) 5'- [phos] CTGTCTCTTATACACATCT -3' 3’- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTAGAGGGATATCACTCAGCATAATTAGAGTAC -5’ Fragments created after transposition: 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATC -3' 3'- CTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: NNNNN------ CTGTCTCTTATACACATC -3' 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 3'- CTACACATATTCTCTGTC ------NNNN After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN 3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ After IVT, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). Can also try adding adapter to 3'OH end with T4 RNA ligase. All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7. RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN -3' RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors (primer 1, from nextera) 5'- AATGATACGGCGACCACCGA -3' (second strand synthesis)(T7tspn-sss) 5'-[AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGA -3' RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' (illuminia bPCR adaptor) (T7tspn-fss) 3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' (first stand synthesis) 3'- AGCATACGGCAGAAGACGAAC -5' (primer 2, from nextera) read primers/amplification etc (only need 1 since only 1 5' end sequence) (T7tspn-Read2) (illuminia bPCR adaptor) 3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5' 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3’ [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' 5'- AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (illuminia bPCR adaptor) (T7tspn-Read1-2) (index read primer) 5'- TTTTTTTTTTTTTTTTTCTGGC -3' (T7tspn-indx)
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 adaptors to 3' ends and PCR amplification
10. size selection.
ordered/to order
(T7tspn-top) 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ (T7tspn-bot) 5'- /5phos/CTGTCTCTTATACACATCT -3' (T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' For using with(T7-top2) Need 5+ NT upstream of T7 for max efficiency (T7-top2) 5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ (T7-fss) 3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' (T7-fss-shrt) 3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5' (T7-sss) 5'-[AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGA -3' (T7-Nprimer1) 5'- AATGATACGGCGACCACCGA -3' (T7-Nprimer2) 3'- AGCATACGGCAGAAGACGAAC -5' (T7tspn-Read1-2) 5'- AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (T7tspn-Read2) 3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5' (T7tspn-indx) 5'- TTTTTTTTTTTTTTTTTCTGGC -3'
left over notes
Second adaptor 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]