Beads-oligo-dT-seqB (ChemGenes, cat. no. Macosko2011-10 B): |--5'- TTTTTTT
Nextera-R1-rc-polyA: 5'-/phos/
Nextera-R1-bk: 5'-/phos/
Nextera-Ad2-bk: 5'-/phos
Template Switching Oligo (TSO): 5'-
TSO PCR: 5'-
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'-
P5xx-TSO: 5'-
Ad2.x (this is basically Nextera N7xx): 5'-
Read1CustomSeqB: 5'- GCCTGTCCGCGG
Nextera-R1 primer: 5'-
Nextera-R2 primer: 5'-
RNA i7 index sequencing primer: 5'-
i5 index sequencing primer: 5'- GT
mRNA (unaffected): 5'- XXXXXXXXXXXX...XXXXXXXXXXXX(pA) Open chromatin DNA product 1 (s5 at both ends:) 5'- TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT -5' Open chromatin DNA product 2 (s7 at both ends): 5'-GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' Open chromatin DNA product 3 (different ends): 5'-TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
mRNA: |--5'- TTTTTTT AAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT) (pA)XXXXXXXXXXXX...XXXXXXXXXXXX -5' Open chromatin DNA product 1: |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT 3'- (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC p XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT -5' Open chromatin DNA product 3: |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT 3'- (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC p XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
mRNA (the terminal transferase activity of MMLV adds extra Cs, which allows TSO to be incorporated): |--5'- TTTTTTT AAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)XXX...XXXCCC-----> <-------(pA)XXX...XXXGGGTAAGTGAGACGCAACTATGGTGACGAA -5' Open chromatin DNA product 1: |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXX----->CTGTCTCTTATACACATCT ------> <---(pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC p <----XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT -5' Open chromatin DNA product 3: |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXX----->CTGTCTCTTATACACATCT ------> <---(pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC p <----XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
mRNA (the terminal transferase activity of MMLV adds extra Cs, which allows TSO to be incorporated): |--5'- TTTTTTT AAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)XXX...XXXCCCATTCACTCTGCGTTGATACCACTGCTT AAAAAAATTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)XXX...XXXGGGTAAGTGAGACGCAACTATGGTGACGAA -5' Open chromatin DNA product 1 (the gap between XXXXXX and ME will be closed by the T7 ligase): Gap ↓ |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT GACGCTGCCGACGA -3' 3'- AAAAAAATTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT -5' ⇩⇩ Gap filled by T7 Open chromatin DNA product 3 (the gap between XXXXXX and ME will be closed by the T7 ligase): Gap ↓ |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC -3' 3'- AAAAAAATTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' ⇩⇩ Gap filled by T7
mRNA (TSO-PCR single primer amplification): 5'- AAGCAGTGGTATCAACGCAGAGT ----------> |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)XXX...XXXCCCATTCACTCTGCGTTGATACCACTGCTT AAAAAAATTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)XXX...XXXGGGTAAGTGAGACGCAACTATGGTGACGAA -5' <----------TGAGACGCAACTATGGTGACGAA -5' Open chromatin DNA (only product 3 will be amplified): 5'-AAGCAGTGGTATCAACGCAGAGT ----------> |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC -3' 3'- AAAAAAATTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' <----------GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
mRNA: 5'- AAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)XXX...XXXCCCATTCACTCTGCGTTGATACCACTGCTT -3' 3'-TTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)XXX...XXXGGGTAAGTGAGACGCAACTATGGTGACGAA -5' Open chromatin DNA: 5'-AAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC -3' 3'-TTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
mRNA: Step 1: Amplify with TSO-PCR again, 0.6X SPRI purification and perform tagmentation with Nextera XT. There will be a few different product after tagmentation. Only amplifiable fragment is shown here. Check the Drop-seq page for more details if you want to know exactly what happens here. 5'- AAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)XXX...XXXCTGTCTCTTATACACATCT 3'-TTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' Step 2: Amplify the tagmented product with P5xx-TSO and Ad2.x primers: 5'-AATGATACGGCGACCACCGAGATCTACAC [8-bp i5 index]GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT AC----------> 5'-AAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)XXX...XXXCTGTCTCTTATACACATCT 3'-TTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' <----------GGCTCGGGTGCTCTG [8-bp i7 index]TAGAGCATACGGCAGAAGACGAAC -5' Open chromatin DNA (this is the easy part, directly amplify with P5xx-TSO and Ad2.x primers): 5'-AATGATACGGCGACCACCGAGATCTACAC [8-bp i5 index]GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT AC----------> 5'-AAGCAGTGGTATCAACGCAGAGT AC[12-bp cell barcode] [8-bp UMI] (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC -3' 3'-TTCGTCACCATAGTTGCGTCTCA TG[12-bp cell barcode] [8-bp UMI] (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' <----------GGCTCGGGTGCTCTG [8-bp i7 index]TAGAGCATACGGCAGAAGACGAAC -5'
mRNA: 5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT ACNNNNNNNNNNNN NNNNNNNN (dT)XXX...XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCA TGNNNNNNNNNNNN NNNNNNNN (pA)XXX...XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 i5ISPCR/TSO 12bp cell 8bp cDNAME s7 i7Illumina P7 barcode UMI Open chromatin: 5'-AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT ACNNNNNNNNNNNN NNNNNNNN (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXX...XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCA TGNNNNNNNNNNNN NNNNNNNN (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 i5ISPCR/TSO 12bp cell 8bp s5 ME gDNAME s7 i7Illumina P7 barcode UMI
mRNA: 5'- GCCTGTCCGCGG AAGCAGTGGTATCAACGCAGAGT AC-------------------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCA TGNNNNNNNNNNNN NNNNNNNN (pA)XXX...XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5' Open chromatin: 5'- GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT AC-------------------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCA TGNNNNNNNNNNNN NNNNNNNN (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
mRNA: 5'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCA TGNNNNNNNNNNNN NNNNNNNN (pA)XXX...XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5' Open chromatin: 5'-TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCA TGNNNNNNNNNNNN NNNNNNNN (pA)AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
mRNA: 5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT ACNNNNNNNNNNNN NNNNNNNN (dT)XXX...XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <-------CGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCA TG -5' Open chromatin: 5'-AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT ACNNNNNNNNNNNN NNNNNNNN (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXX...XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG <-------CGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCA TG -5'
mRNA: 5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT ACNNNNNNNNNNNN NNNNNNNN (dT)XXX...XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <-------GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' Open chromatin: 5'-AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT ACNNNNNNNNNNNN NNNNNNNN (dT)TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXX...XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG <-------GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'