scifi-ATAC-seq

The idea of scifi-ATAC-seq is very similar to dsciATAC-seq and txci-ATAC-seq. It works on the 10X Genomics Single Cell ATAC platform, but it does require custom sequencing primer and recipe. The procedure was based on the information from the preprint.

Adapter and primer sequences:

* The exact sequences of barcodes and other oligos can be found in their Supplementary Table S1

10x Genomics Beads-oligo: |--5'- AATGATACGGCGACCACCGAGATCTACAC[16-bp GEM barcode]TCGTCGGCAGCGTC -3'

Tn5-ME-A: 5'- TCGTCGGCAGCGTCGATATGTGATAATGAGGAC[5-bp Tn5 index A]AGATGTGTATAAGAGACAG -3'

Tn5-ME-B: 5'- GTCTCGTGGGCTCGGTGAATGTGTAGAAGACAGA[5-bp Tn5 index B]AGATGTGTATAAGAGACAG -3'

Tn5ME-bottom: 5'-/phos/ CTGTCTCTTATACACATCT -3'

Nextera Tn5 binding site (19-bp Mosaic End (ME)): 5'- AGATGTGTATAAGAGACAG -3'

Nextera S5xx primer entry point (s5): 5'- TCGTCGGCAGCGTC -3'

Nextera N7xx primer entry point (s7): 5'- GTCTCGTGGGCTCGG -3'

i7 Sample Index Plate N, Set A (PN-3000262): 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp sample index]GTCTCGTGGGCTCGG -3'

Custom Read 1 sequencing primer (1_Read1): 5'- TCGTCGGCAGCGTCGATATGTGATAATGAGGAC -3'

Custom i7 index sequencing primer (2_Index1): 5'- TCTGTCTTCTACACATTCACCGAGCCCACGAGAC -3'

Custom i5 index sequencing primer (3_Index2): 5'- GTCCTCATTATCACATATCGACGCTGCCGACGA -3'

Custom Read 2 sequencing primer (4_Read2): 5'- GTCTCGTGGGCTCGGTGAATGTGTAGAAGACAGA -3'

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Step-by-step library generation:

(1) Anneal "Tn5ME-bottom+Tn5-ME-A" and "Tn5ME-bottom+Tn5-ME-B" and use those to assemble the indexed Tn5 transposomes:

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


Product 1 (s5 at both ends, not amplifiable due to semi-suppressiev PCR):

5'- TCGTCGGCAGCGTCGATATGTGATAATGAGGAC[5-bp Tn5 index A]AGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                                       TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA[5-bp Tn5 index A]CAGGAGTAATAGTGTATAGCTGCGACGGCTGCT -5'


Product 2 (s7 at both ends, not amplifiable due to semi-suppressiev PCR):

5'- GTCTCGTGGGCTCGGTGAATGTGTAGAAGACAGA[5-bp Tn5 index B]AGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                                        TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA[5-bp Tn5 index B]AGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTG -5'


Product 3 (different ends, amplifiable):

5'- TCGTCGGCAGCGTCGATATGTGATAATGAGGAC[5-bp Tn5 index A]AGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                                       TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA[5-bp Tn5 index B]AGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTG -5'

(3) Load to 10X machine for GEM barcode addition:


Gap fill-in (72 C, 5 mins):

5'- TCGTCGGCAGCGTCGATATGTGATAATGAGGAC[5-bp Tn5 index A]AGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCT[5-bp Tn5 index B]TCTGTCTTCTACACATTCACCGAGCCCACGAGAC -3'
3'- AGCAGCCGTCGCAGCTATACACTATTACTCCTG[5-bp Tn5 index A]TCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGA[5-bp Tn5 index B]AGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTG -5'


Single-primer linear PCR for GEM barcode addition:

|--5'- AATGATACGGCGACCACCGAGATCTACAC[16-bp GEM barcode]TCGTCGGCAGCGTC---------->
                                                   5'- TCGTCGGCAGCGTCGATATGTGATAATGAGGAC[5-bp Tn5 index A]AGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCT[5-bp Tn5 index B]TCTGTCTTCTACACATTCACCGAGCCCACGAGAC -3'
                                                   3'- AGCAGCCGTCGCAGCTATACACTATTACTCCTG[5-bp Tn5 index A]TCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGA[5-bp Tn5 index B]AGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTG -5'

(4) Break emulsion and DNA purification. This is the product from above:


5'- AATGATACGGCGACCACCGAGATCTACAC[16-bp GEM barcode]TCGTCGGCAGCGTCGATATGTGATAATGAGGAC[5-bp Tn5 index A]AGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCT[5-bp Tn5 index B]TCTGTCTTCTACACATTCACCGAGCCCACGAGAC
    TTACTATGCCGCTGGTGGCTCTAGATGTG[16-bp GEM barcode]AGCAGCCGTCGCAGCTATACACTATTACTCCTG[5-bp Tn5 index A]TCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGA[5-bp Tn5 index B]AGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTG -5'

(5) Sample Index PCR using Illumina P5 and i7 Sample Index Plate N (PN-3000262):


5'- AATGATACGGCGACCACCGAGATCTACAC------------------->
5'- AATGATACGGCGACCACCGAGATCTACAC[16-bp GEM barcode]TCGTCGGCAGCGTCGATATGTGATAATGAGGAC[5-bp Tn5 index A]AGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCT[5-bp Tn5 index B]TCTGTCTTCTACACATTCACCGAGCCCACGAGAC
    TTACTATGCCGCTGGTGGCTCTAGATGTG[16-bp GEM barcode]AGCAGCCGTCGCAGCTATACACTATTACTCCTG[5-bp Tn5 index A]TCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGA[5-bp Tn5 index B]AGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTG -5'
                                                                                                                                                                          <----------------GGCTCGGGTGCTCTG[8-bp sample index]TAGAGCATACGGCAGAAGACGAAC -5'

(6) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNNNNNNNTCGTCGGCAGCGTCGATATGTGATAATGAGGACNNNNNAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTNNNNNTCTGTCTTCTACACATTCACCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
    TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNNNNNNNAGCAGCCGTCGCAGCTATACACTATTACTCCTGNNNNNTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGANNNNNAGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
             Illumina P5              16 bp           s5                          5 bp        ME            gDNA          ME          5 bp                          s7         8 bp        Illumina P7
                                   GEM barcode                                 Tn5 Index A                                        Tn5 Index B                             sample index

Library sequencing:

(1) Add 1_Read1 sequencing primer to sequence the first read (at least 74 cycles, bottom strand as template, the first 5 bp are the Tn5 Index A, the last >50 bp are gDNA, the middle 19 bp are ME):


                                             5'- TCGTCGGCAGCGTCGATATGTGATAATGAGGAC------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNNNNNNNAGCAGCCGTCGCAGCTATACACTATTACTCCTGNNNNNTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGANNNNNAGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add 2_Index1 sequencing primer to sequence sample index (i7) (bottom strand as template, 8 cycles):


                                                                                                                                       5'- TCTGTCTTCTACACATTCACCGAGCCCACGAGAC------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNNNNNNNAGCAGCCGTCGCAGCTATACACTATTACTCCTGNNNNNTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGANNNNNAGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add 3_Index2 sequencing primer to sequence the second index (i5) (top strand as template, 16 cycles, this is the GEM barcode):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNNNNNNNTCGTCGGCAGCGTCGATATGTGATAATGAGGACNNNNNAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTNNNNNTCTGTCTTCTACACATTCACCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
                                 <---------------AGCAGCCGTCGCAGCTATACACTATTACTCCTG -5'

(4) Add 4_Read2 sequencing primer to sequence the second read (top strand as template, at least 74 cycles, the first 5 bp are the Tn5 Index B, the last >50 bp are gDNA, the middle 19 bp are ME):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNNNNNNNTCGTCGGCAGCGTCGATATGTGATAATGAGGACNNNNNAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTNNNNNTCTGTCTTCTACACATTCACCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
                                                                                                               <---------------------------AGACAGAAGATGTGTAAGTGGCTCGGGTGCTCTG -5'