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===Method using PCR for addition of T7 polymerase=== Protocol: *1. tagmentation with nextera kit *2. Addition of T7 adaptors, (adaptor 1, adaptor 2) with PCR amplification *3. In vitro transcription with T7, overnight for 16 hours. *4. RNA fragmentation and then possibly A-tail the RNA *5. cDNA synthesis *6. PolyA addition with Terminal Transferase (if haven't A-tailed the RNA) *7. Addition of bPCR lawn adaptors with primer mix of sequences for (orange, blue, polyT addition) *8. now ready for input into the sequencer with the combined nextera read primer 1, and nextera read primer 2. Adaptor 2 barcode (illuminia bPCR adaptor 2) Blue Orange 3'- GACTCGCCCGACCGTTCCGTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' 5'- GCCT CCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' 3'- CGGA GGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]CCCTCGCGCCATCAG -3' Adaptor 1 (illuminia bPCR adaptor) Adaptor 2 <-- T7 Blue Orange 3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTACATC -5' 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' 5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGT -3' Adaptor 1 T7 --> 5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGT -3' 3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTACATC -5' Adaptor 1 T7 --> Adaptor 2 <-- T7 After PCR with Adatper 1/Adaptor 2 with T7(and cDNA generation) Blue Orange <-- T7 5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCT[CCCTATAGTGAGTCGTATTAA]CTCATGTAG -3' 3'- GATGTACTC[AATTATGCTGAGTGATATCCC]TCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTACATC -5' T7 --> RNA synthesized by T7, then RNA fragmentation, RNA A-tailing [http://products.invitrogen.com/ivgn/product/AM1350 Ambion PolyA tailing kit], then cDNA synthesis. Blue 5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN -3' RNA 5'- GGG AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN -3' Orange 5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN -3' RNA 5'- GGG AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN -3' Adaptors for first/second strand cDNA synthesis (after polyA tailing of RNA frags???): Blue (second strand synthesis) 5'- GGGAGCCTCCCTCGCGCCA -3' RNA 5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' 3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5' (first strand synthesis) Orange (second strand synthesis) 5'- GGGAGCCTTGCCAGCCCGC -3' RNA 5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' 3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5' (first strand synthesis) lawn 1 adaptors added using orange and blue sequences, and lawn 2 adaptors added to polyA tail Blue (illuminia bPCR adaptor) 3'- V TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' 5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3' 3'- CCCTCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTT -5' 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTCCCTCGCGCCATCAG -3' (illuminia bPCR adaptor, sequence modified) (illuminia bPCR adaptor) Orange 3'- V TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' 5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3' 3'- CCCTCGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTT -5' 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGT -3' (illuminia bPCR adaptor, sequence modified) Now Ready for sequencing: Blue (nextera read primer 1) 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3' 3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' (illuminia bPCR adaptor) (illuminia bPCR adaptor) Orange (nextera read primer 2) 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3' 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3' 3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' (illuminia bPCR adaptor) (illuminia bPCR adaptor) Index read primer for both Orange and Blue: 5'- AAAAAAAAAAAACTGGC -3' (index read primer) *PCR amplificiation with both bPCR adaptors *nextera read 1 and nextera read 2 can be combined in the sequencing reaction, then all combinations will be sequenced: **blue orange **orange blue **orange orange **blue blue
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