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===modification of Shendure protocol with ME-t7=== *modification and addition of T7 in place of adaptor sequences **see [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-2-6 T7 information etc] **first G in GGG is the +1 site **at least 5 nucleotides needed before start of sequence to ensure correct binding *ME sequence + Blue or orange sequence = length of nextera read primer 1, nextera read primer 2. *Adaptor 1 to Blue primer, Adaptor 2 to Orange primer. Bother will have T7 on them, thus both ends will always be tagged. **flipped orientations results in the reversed strandedness of T7 synthesis direction. Protocol: *1. tagmentation with nextera kit *2. Partially denature at 72C (5-10C higher than the Tm for ME sequences) *3. Anneal a ME-T7 adaptor. make it specific for orange/blue too? *4. Gap filling and ligation. (T4 DNAP/Ampligase as in Shendure protocol) *5. IVT and conversion to sequencing libraries. Summary: T7 description: +1 CATGAGA[TTAATACGACTCACTATA|G|GGAGA] [end sequence for optimal binding] T7 --> [front sequence for optimal binding] After Nextera tagmentation: Orange Blue 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' Blue Orange 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Orange Orange 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Blue Blue 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' Partially denaturing at 72C, higher temp than Tm for ME sequence: Orange Blue 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- -3' 3'- ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' Blue Orange 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- -3' 3'- ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Orange Orange 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- -3' 3'- ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Blue Blue 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- -3' 3'- ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' Anneal ME-T7 adaptor (add more sequence specific to orange/blue?) Orange Blue <-- T7 Comp (ME-T7-RC) 5'- GCCTTGCCAGCCCGCTCAG AGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' X XXXX XXXXXX XXXXX XXXXXXXXXX X X XX X 3'-GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGA GACTACCGCGCTCCCTCCG -5' (ME-T7-RC) T7 comp --> Fill-in and ligation to append ME-T7 adaptors. (shendure used T4 DNAP and ampligase) Orange Blue <-- T7 Comp 5'- GCCTTGCCAGCCCGCTCAG AGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' X XXXX XXXXXX XXXXX XXXXXXXXXX X X XX X 3'-GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGA GACTACCGCGCTCCCTCCG -5' T7 comp --> Denature, anneal with T7 primer for second strand synthesis (taq polymerase): Can also try IVT without second strand synthesis, (reports possibly saying T7 can initiate without needing a double stranded template). Orange <-- T7 Comp 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' 3'- [AGAGGGATATCACTCAGCATAATT]AGAGTAC -5' (T7prmr-sss) Blue 5'- CATGAGA[TTAATACGACTCACTATAGGGAGA] -3' (T7prmr-sss) 3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' T7 comp --> After second strand synthesis: Orange <-- T7 Comp 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' 3'- CGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGA[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5' Blue 5'- CATGAGA[TTAATACGACTCACTATAGGGAGA]AGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3' 3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' T7 comp --> After RNA generation from IVT. addition of lawn adapters (then fragmentation and polyA addition???): Can also try adding adapter to 3'OH end with T4 RNA ligase. primers for ends can also be used, same primers used in nextera. (could update with polyA addition sometime, which will probably be done instead) (2nd strand synthesis)(bPCR adaptor) 5'- AATGATACGGCGACCACCGA -3' (primer 1, from nextera) 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG -3' Blue RNA 5'- GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3' (bPCR adaptor) 3'- TAGAGACTACCGCGCTCCCTCCGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' 3'- AGCATACGGCAGAAGACGAAC -5' (first strand synthesis-B) (primer 2, from nextera) (2nd strand synthesis)(bPCR adaptor) 5'- AATGATACGGCGACCACCGA -3' (primer 1, from nextera) 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG -3' Orange RNA 5-' GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' (bPCR adaptor) 3'- TAGAGACTCGCCCGACCGTTCCGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' 3'- AGCATACGGCAGAAGACGAAC -5' (first strand synthesis-O) (primer 2, from nextera) now ready for sequencing: Blue (read 1 primer) 5'- GGGAGAAGATGTGTATAAGAGACAG -3' 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGCGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3' 3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCTTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' 3'- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCC -5' (read primer 2) 5'- TGATGGCGCGAGGGAGGCGACCG -3' (index read primer 1) Orange (read 1 primer) 5'- GGGAGAAGATGTGTATAAGAGACAG -3' 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCCGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3' 3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCTTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG (read primer 2) 5'- CTGAGCGGGCTGGCAAGGCCGACCG -3' (index read primer 2)
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