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===Single cell transposon design=== sc-ME-top 5'- /5Phos/GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-bottom 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC]/5Phos/ -5' 5'- /5Phos/[CTGTCTCTTATACACATCT]CTGATGGCGC|GAGGGAGGAT -3' sc-T7-R1-IdxXX 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC] -3' sc-T7-R1-Idx49 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][CTGTGT][ATCCTCCCTC] -3' sc-T7-R1-Idx50 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][ACCTTT][ATCCTCCCTC] -3' sc-T7-R1-Idx51 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TATCGC][ATCCTCCCTC] -3' sc-T7-R1-Idx52 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][GACACG][ATCCTCCCTC] -3' sc-T7-R1-Idx53 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TTCTAC][ATCCTCCCTC] -3' sc-T7-R1-Idx54 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][ACGTCC][ATCCTCCCTC] -3' sc-T7-R1-Idx55 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TCGACT][ATCCTCCCTC] -3' sc-T7-R1-Idx56 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TCAGAC][ATCCTCCCTC] -3' sc-T7-R1-Idx57 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TCCTTC][ATCCTCCCTC] -3' sc-T7-R1-Idx58 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][CAGCAT][ATCCTCCCTC] -3' sc-T7-R1-Idx59 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][GATAGA][ATCCTCCCTC] -3' sc-T7-R1-Idx60 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][ACAGAT][ATCCTCCCTC] -3' sc-T7-R1-Idx61 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][CTCTAT][ATCCTCCCTC] -3' Complete sequence of transposome after ligation T7 consensus sequence (27) N2 Index seq (26) bc(6) for ligation/R1 (20) ME end (19) 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC|GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' After annealing sc-ME-annealed 5'- /5Phos/GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-top 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC]/5Phos/ -5' sc-ME-bottom After transposition (fragment from two adjacent insertions) 5'- /5Phos/|GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT -3' 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG/5Phos/ -5' Single insertion 5'- NN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT /5Phos/|GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN -3' 3'- NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG/5Phos/ TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NN -5' After ligation with T4 (amp ligase?) (single insertion) 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC|GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN -3' sc-ME-top + sc-T7-R1-Idx49 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NN -5' sc-ME-bottom After gap fill in/extension with taq2x 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- TTTAATTATGCTGAGTGATATCCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' After IVT, RNA generated RNA 5'- GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' After single strand cDNA synthesis to form cDNA/RNA hybrid RNA 5'- GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer. Extension with BST or klenow exo- or DNAP1 or Taq2x DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG -3' (sc-SSS-prmr)(NEB Tm=78C) cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' Now have double stranded DNA, which should be long DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -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 for 3' end 5'- /5Phos/CTGTCTCTTATACACATCT -3' (sc-NXTA_ME_bot, "pMENTS") 3'- GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5' (sc-ILA-adaptor) 3' ILA adaptor 3'- TCTACACATATTCTCTGTC/5Phos/ (sc-NXTA_ME_bot, "pMENTS") 5'- [CAAGCAGAAGACGGCATACGAGAT]CGAGATGTGTATAAGAGACAG -3' (sc-ILA-adaptor) 3' ILA adaptor After custom nextera tagmentation: Now have double stranded DNA, which should be long DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCT -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGCTAGAGCATACGGCAGAAGACGAAC -5' After fill in: DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCGATCTCGTATGCCGTCTTCTGCTTG -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGCTAGAGCATACGGCAGAAGACGAAC -5' Amplification: ILA 5' adaptor 5β- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGA sc-T7-5'-PCR1 (comp part (NEB Tm=72), all (NEB Tm=83C) 5β- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCA sc-T7-5'-PCR2 (comp part (Tm=68), all (Tm=81C)) ILA 3' adaptor DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCG[ATCTCGTATGCCGTCTTCTGCTTG] -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5' 3'- AGCATACGGCAGAAGACGAAC -5' (sc-NXTA_3'-P2) (NEB Tm=65C) 3'- GC[TAGAGCATACGGCAGAAGACGAAC -5 (sc-NXTA_P2-3'_E) (NEB Tm=70C) Read primers (N2 Index read) T7tspn-Read1 (Tm=75.80) (same as NXTA R1) ILA 5' adaptor AAGAGCGGTTCAGCAGGAATGCCGAG TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG ILA 3' adaptor 5β- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCG[ATCTCGTATGCCGTCTTCTGCTTG] -3' 3'- [TTACTATGCCGCTGGTGGCT]CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5' primers ordered sc-ME-top 5'- /5Phos/GCGCCATCAGAGATGTGTATAAGAGACAG -3' sc-ME-bottom 5'- /5Phos/CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGAT -3' sc-SSS-prmr /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG -3' sc-ILA-adaptor CAAGCAGAAGACGGCATACGAGATCGAGATGTGTATAAGAGACAG sc-NXTA_ME_bot /5Phos/CTGTCTCTTATACACATCT sc-T7-5'-PCR1 AATGATACGGCGACCACCGAGGGAGAAAGAGCGGTTCAGCAGGA sc-T7-5'-PCR2 AATGATACGGCGACCACCGAGGGAGAAAGAGCGGTTCAGCA sc-NXTA-3'_P2 CAAGCAGAAGACGGCATACGA sc-NXTA-3'_P2_E CAAGCAGAAGACGGCATACGAGATCG sc-T7-R1-Idx49 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGCTGTGTATCCTCCCTC -3' sc-T7-R1-Idx50 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGACCTTTATCCTCCCTC -3' sc-T7-R1-Idx51 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTATCGCATCCTCCCTC -3' sc-T7-R1-Idx52 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGGACACGATCCTCCCTC -3' sc-T7-R1-Idx53 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTTCTACATCCTCCCTC -3' sc-T7-R1-Idx54 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGACGTCCATCCTCCCTC -3' sc-T7-R1-Idx55 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTCGACTATCCTCCCTC -3' sc-T7-R1-Idx56 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTCAGACATCCTCCCTC -3' sc-T7-R1-Idx57 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTCCTTCATCCTCCCTC -3' sc-T7-R1-Idx58 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGCAGCATATCCTCCCTC -3' sc-T7-R1-Idx59 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGGATAGAATCCTCCCTC -3' sc-T7-R1-Idx60 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGACAGATATCCTCCCTC -3' sc-T7-R1-Idx61 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGCTCTATATCCTCCCTC -3'
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