It has been previously demonstrated that Tn5 transposase-mediated tagmentation contains two stages: (1) a tagging stage where the Tn5 transposome binds to DNA, and (2) a fragmentation stage where the Tn5 transposase is released from DNA using heat or denaturing agents, such as sodium dodecyl sulfate (SDS). As the Tn5 tagging does not fragment DNA, the nuclei would remain intact after incubation with the Tn5 transposome in an bulk ATAC-seq experiment. Both plate_scATAC-seq and Pi-ATAC-seq utilise this property of Tn5 tagmentation, and perform bulk tagging upfront, followed by FACS sorting of single nuclei into wells for library construction. FACS index sorting can also be reported. These methods are simple to follow and work robustly. Since each cells are processed separately, the library indices are single cell barcodes.
Nextera Tn5 binding site (19-bp Mosaic End (ME)): 5'-
Nextera N/S5xx primer entry point (s5): 5'-
Nextera N7xx primer entry point (s7): 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
Nextera (XT) N/S5xx Index primer: 5'-
Nextera (XT) N7xx Index primer: 5'-
Read 1 sequencing primer: 5'-
Index 1 sequencing primer: 5'-
Index 2 sequencing priemr: 5'-
Read 2 sequencing primer: 5'-
N/S502 : CTCTCTAT N/S503 : TATCCTCT N/S505 : GTAAGGAG N/S506 : ACTGCATA N/S507 : AAGGAGTA N/S508 : CTAAGCCT N/S510 : CGTCTAAT N/S511 : TCTCTCCG N/S513 : TCGACTAG N/S515 : TTCTAGCT N/S516 : CCTAGAGT N/S517 : GCGTAAGA N/S518 : CTATTAAG N/S520 : AAGGCTAT N/S521 : GAGCCTTA N/S522 : TTATGCGA N701 : TCGCCTTA N702 : CTAGTACG N703 : TTCTGCCT N704 : GCTCAGGA N705 : AGGAGTCC N706 : CATGCCTA N707 : GTAGAGAG N710 : CAGCCTCG N711 : TGCCTCTT N712 : TCCTCTAC N714 : TCATGAGC N715 : CCTGAGAT N716 : TAGCGAGT N718 : GTAGCTCC N719 : TACTACGC N720 : AGGCTCCG N721 : GCAGCGTA N722 : CTGCGCAT N723 : GAGCGCTA N724 : CGCTCAGT N726 : GTCTTAGG N727 : ACTGATCG N728 : TAGCTGCA N729 : GACGTCGA
Product 1 (s5 at both ends, not amplifiable due to semi-suppressiev PCR): 5'- TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT -5' Product 2 (s7 at both ends, not amplifiable due to semi-suppressiev PCR): 5'-GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' Product 3 (different ends, amplifiable): 5'-TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
5'- TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
5'- AATGATACGGCGACCACCGAGATCTACAC [i5]TCGTCGGCAGCGTC ----> 5'-TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' <----GGCTCGGGTGCTCTG [i7]TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNTCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNAGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 i5s5 ME cDNAME s7 i7Illumina P7
5'- TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG ------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNAGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC ------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNAGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNTCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG <-------AGCAGCCGTCGCAG TCTACACATATTCTCTGTC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNTCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG <------GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'