CH-ATAC-seq

CH-ATAC-seq is like sci-ATAC-seq3 that uses combinatorial indexing to achieve single cell chromatin accessibility profiling. They difference is that CH-ATAC-seq uses barcoded Tn5 with to add the first-level barcode. Then subsequent rounds of barcodes are added through hybridisation. The oligo sequences here are taken from the Supplementary Table 1 from the paper, which is designed to work on the BGI system.

Adapter and primer sequences:

Step-by-step library generation

(1) Anneal "Tn5_ME+Tn5_barcode_primer" and "Tn5_ME+Tn5_Primer_C_oligo" and use those to assemble indexed Tn5 transposome:

(2) Bulk nuclei tagging by the indexed Tn5 shown above. There are 3 different products (will create 9 bp gap):

Product 1 (purple at both ends, not amplifiable due to semi-suppressiev PCR, omitted afterwards):

5'- AAGCAGTGGTATCAACGCAGAGT[10-bp Tn5 barcode]AGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                              TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA[10-bp Tn5 barcode]TGAGACGCAACTATGGTGACGAA -5'


Product 2 (orange at both ends, cannot be ligated, omitted afterwards):

5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                   TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'


Product 3 (different ends, the only usable fragment):

5'- AAGCAGTGGTATCAACGCAGAGT[10-bp Tn5 barcode]AGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                              TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(3) Pool nuclei and redistribute to new plates for hybridisation barcode addition:

(3.1) Anneal HY_head_oligo with Barcoded_HY_oligo_{1..768}:

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC[10-bp HY barcode]TTCGTCACCATAGTTGCGTCTCA -5'


(3.2) Hybridise to transposed nuclei and ligate the HY barcode:

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG                  AAGCAGTGGTATCAACGCAGAGT[10-bp Tn5 barcode]AGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC[10-bp HY barcode]TTCGTCACCATAGTTGCGTCTCA -5'               TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(4) Add Block_tail_primer_oligo to block unused HY oligos, pool nuclei, redistribute to new plates and perform gap fill-in:

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG[10-bp HY barcode]AAGCAGTGGTATCAACGCAGAGT[10-bp Tn5 barcode]AGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC
    AGCAGCCGTCGCAGTCTACACATATTCTCTGTC[10-bp HY barcode]TTCGTCACCATAGTTGCGTCTCA[10-bp Tn5 barcode]TCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(5) Lyse cells and use PCR P5 primer + MGI_P7_index_{1..96} primer to amplify the library:

5'- GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTC---------->
                         5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG[10-bp HY barcode]AAGCAGTGGTATCAACGCAGAGT[10-bp Tn5 barcode]AGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC
                             AGCAGCCGTCGCAGTCTACACATATTCTCTGTC[10-bp HY barcode]TTCGTCACCATAGTTGCGTCTCA[10-bp Tn5 barcode]TCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                                                                               <---------GGCTCGGGTGCTCTG[10-bp i7]CTTCTGTTGTTGAGGAACCGAGTGT -5'

(6) Final library structure:

5'- GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNAAGCAGTGGTATCAACGCAGAGTNNNNNNNNNNAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNNNGAAGACAACAACTCCTTGGCTCACA -3'
3'- CTTGCTGTACCGATGCTAGGCTGAAAGCAGCCGTCGCAGTCTACACATATTCTCTGTCNNNNNNNNNNTTCGTCACCATAGTTGCGTCTCANNNNNNNNNNTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNNNCTTCTGTTGTTGAGGAACCGAGTGT -5'
              MGI P5               s5             ME            10-bp         HY linker          10-bp           ME           gDNA          ME                s7        10-bp i7          MGI P7
                                                             HY barcode                       Tn5 barcode

Library sequencing (not sure how MGI sequencing works, so this section is just based on guesses):

(1) Add Read 1 sequencing primer to sequence the first read (bottom strand as template, 100 cycles, with dark cycles from 11-33 and 44-62):

                         5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG----------ooooooooooooooooooooooo----------ooooooooooooooooooo--->
3'- CTTGCTGTACCGATGCTAGGCTGAAAGCAGCCGTCGCAGTCTACACATATTCTCTGTCNNNNNNNNNNTTCGTCACCATAGTTGCGTCTCANNNNNNNNNNTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNNNCTTCTGTTGTTGAGGAACCGAGTGT -5'

(2) Add Index 1 sequencing primer to sequence the i7 index (bottom strand as template, 10 cycles):

                                                                                                                                 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC--------->
3'- CTTGCTGTACCGATGCTAGGCTGAAAGCAGCCGTCGCAGTCTACACATATTCTCTGTCNNNNNNNNNNTTCGTCACCATAGTTGCGTCTCANNNNNNNNNNTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNNNCTTCTGTTGTTGAGGAACCGAGTGT -5'

(3) Add Read 2 sequencing primer to sequence the second read (top strand as template, 100 cycles):

5'- GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNAAGCAGTGGTATCAACGCAGAGTNNNNNNNNNNAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNNNGAAGACAACAACTCCTTGGCTCACA -3'
                                                                                                                             <-------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'