THS-seq, originally published by Sos et al., uses similar principles as ATAC-seq to investigate chromatin accessibility. The key difference is that in THS-seq, a Tn5 transoposome homodimer loaded with T7 promoter sequence is used for the tagmentation, and the resulting fragments are amplified by in vitro transcription. In this way, every Tn5 cutting event is theoretically amplifiable, whereas in the widely used Illumina Tn5 transposomes only 50% of fragments with distinct adapters can be amplified.
The scTHS-seq takes the combinatorial indexing strategy to make the original THS-seq method to work at the single cell level.
scT7_r5{001..384}_i5_top: 5'- AAT
* There are a total of 384 barcoded scT7_r5 sequences. For the full list, check Supplementary Table 12. from the publication.
nXTv2_i7_top: 5'-
ME_bottom: 5'- /Phos/-
sss_scnXTv2: 5'-
T7 promoter: 5'-
Random hexamer for reverse transcription: 5'-
scT7_S5xx: 5'-
* There are a total of 16 scT7_S5xx sequences. For the full list, check Supplementary Table 12. from the publication.
Nextera (XT) N7xx Index primer: 5'-
nXTv2_read1 sequencing primer: 5'-
nXTv2_i7_index_read sequencing primer: 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
Since it is a Tn5 homodimer, there is only one product: 5'- AAT TAATACGACTCACTATAGGG AGATCCACGCGC [6-bp Tn5 barcode] TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT -3' 3'-TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT [6-bp Tn5 barcode] CGCGCACCTAGA GGGATATCACTCAGCATAAT TAA -5'
5'- AAT TAATACGACTCACTATAGGG AGATCCACGCGC [6-bp Tn5 barcode] TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT GACGCTGCCGACGA [6-bp Tn5 barcode] GCGCGTGGATCT CCCTATAGTGAGTCGTATTA ATT -3' 3'- TTAATTATGCTGAGTGATATCCC TCTAGGTGCGCG [6-bp Tn5 barcode] AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT [6-bp Tn5 barcode] CGCGCACCTAGA GGGATATCACTCAGCATAAT TAA -5'
IVT starts from here ↱ 5'- AAT TAATACGACTCACTATAGGG AGATCCACGCGC [6-bp Tn5 barcode] TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT GACGCTGCCGACGA [6-bp Tn5 barcode] GCGCGTGGATCT CCCTATAGTGAGTCGTATTA ATT -3' 3'- TTAATTATGCTGAGTGATATCCC TCTAGGTGCGCG [6-bp Tn5 barcode] AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT [6-bp Tn5 barcode] CGCGCACCTAGA GGGATATCACTCAGCATAAT TAA -5' ↵ IVT starts from here
5'- G AGAUCCACGCGC [6-bp Un5 barcode] UCGUCGGCAGCGUC AGAUGUGUAUAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCUGUCUCUUAUACACAUCU GACGCUGCCGACGA [6-bp Un5 barcode] GCGCGUGGAUCU CCCUAUAGUGAGUCGUAUUA AUU -3'
5'- G AGAUCCACGCGC [6-bp Un5 barcode] UCGUCGGCAGCGUC AGAUGUGUAUAAGAGACAG XXXXXXXXXXXX...XXXXXXXXXXXXCUGUCUCUUAUACACAUCU GACGCUGCCGACGA [6-bp Un5 barcode] GCGCGUGGAUCU CCCUAUAGUGAGUCGUAUUA AUU -3' <----------NNNNNN[maybe there is an overhang] -5'
5'- G AGAUCCACGCGC [6-bp Un5 barcode] UCGUCGGCAGCGUC AGAUGUGUAUAAGAGACAG XXX...XXX[maybe there is an overhang] -3' 3'-C TCTAGGTGCGCG [6-bp Tn5 barcode] AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXX[maybe there is an overhang] -5'
3'- C TCTAGGTGCGCG [6-bp Tn5 barcode] AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXX[maybe there is an overhang] -5'
5'- GGG AGATCCACGCGC --------------------> 3'-C TCTAGGTGCGCG [6-bp Tn5 barcode] AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXX[maybe there is an overhang] -5'
5'- GGG AGATCCACGCGC [6-bp Tn5 barcode] TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXX...XXX[maybe there is an overhang] -3' 3'-CCC TCTAGGTGCGCG [6-bp Tn5 barcode] AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXX[maybe there is an overhang] -5'
Product 1 (s7 at both ends, not amplifiable due to pimers used, see the next step): 5'- GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' Product 2 (not amplifiable due to pimers used, see the next step): 5'-GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXX[maybe there is an overhang] -3'TCTACACATATTCTCTGTC XXX...XXX[maybe there is an overhang] -5' Product 3 (different adapters at the two ends, this will be amplified in the next step): 5'-GGG AGATCCACGCGC [6-bp Tn5 barcode] TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXX...XXXCTGTCTCTTATACACATCT -3' 3'-CCC TCTAGGTGCGCG [6-bp Tn5 barcode] AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
5'- AATGATACGGCGACCACCGAGATCTACAC [8-bp i5 index]GGG AGATCCACGCGC ---------> 5'-GGG AGATCCACGCGC [6-bp Tn5 barcode] TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXX...XXXCTGTCTCTTATACACATCT -3' 3'-CCC TCTAGGTGCGCG [6-bp Tn5 barcode] AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' <----------GGCTCGGGTGCTCTG [8-bp i7 index]TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNGGG AGATCCACGCGC NNNNNN TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXX...XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCCC TCTAGGTGCGCG NNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXX...XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 i5 index6 bp s5 ME gDNAME s7 i7 indexIllumina P7 Tn5 barcode
5'- TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG ------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCCC TCTAGGTGCGCG NNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXX...XXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCCC TCTAGGTGCGCG NNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXX...XXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC -------------------------------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNCCC TCTAGGTGCGCG NNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTC XXXXXX...XXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'