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===Sequences information, Using T7tspn-top2=== Sequences: 5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3β (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: 5β- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3β (T7tspn-top2) 3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot) 5'- [phos] CTGTCTCTTATACACATCT -3' 3β- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTAGAGGGATATCACTCAGCATAATTAGAGTAC -5β Fragments created after transposition: 5β- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5β Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: NNNNN------ CTGTCTCTTATACACATCT -3' 5β- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5β 3'- TCTACACATATTCTCTGTC ------NNNN After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) 5β- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN 3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5β After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT. First G in GGG is the +1 site for the beginning of RNA synthesis for T7. for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator) RNA 5β- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' After single strand cDNA synthesis to form cDNA/RNA hybrid RNA 5β- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer. Extenstion with BST or klenow exo- or DNAP1 or Taq2x DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'(Tm=79) (sss_NPA_prmr) (No Poly A) cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' Now have double stranded DNA, which should be long. DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' normal Nextera 5' end: 5'-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3' ("A-METS" NXTA_ME_A) 3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS") after fill in: 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' PCR amplification: 5'-AATGATACGGCGACCACCGA-3' (NXTA_P1) 5'-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG-3' (NXTA_adpt1) 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' Custom Nextera transposome 5'- /5Phos/CTGTCTCTTATACACATCT -3' (NXTA_ME_BOT, "pMENTS") 3'- GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' (NXTA_ME_B, "B-METS") 3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS") 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3' (NXTA_ME_B, "B-METS") After custom nextera tagmentation: DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3' cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' After fill in: DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Amplification (barcode and ILA adaptor addition): 5β- [AATGATACGGCGACCACCGA]GGG]AGATCCTCCCTCGCGCCATCAGAG T7-top2-PCR-iaf3 (comp part (Tm=77), all (Tm=85C) 5β- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAG T7-top2-PCR-iaf2 (comp part (Tm=73), all (Tm=83C)) X X 5β- [AATGATACGGCGACCACCGA]GATCTCTCCCTCGCGCCATCAGAGAT T7-top2-PCR-iaf (original) (comp region 70C) (whole Tm=83C) X X XXXXX DNA /5Phos/GGG]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' cDNA 3'- CCC]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' 3'- GACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (custom NXTA barcodes) (Tm=86C) 3'- GACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C) 3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2) Read Primers: Using T7-top2-PCR-iaf2 for amp. (Nextera_R1) 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (T7tspn-Read1) (Nextera_Ind index read primer) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' 5'- CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG -3' 5β- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG[XXXXXX]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- [TTACTATGCCGCTGGTGGCT]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' T7tspn-Read1 TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG T7tspn-Read1-long CCGAAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG
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