sci-ATAC-seq / sci-ATAC-seq3


sci-ATAC-seq

The sci-ATAC-seq uses the combinatorial indexing to identify single cells without single cell isolation. Cells can be identified by the unique combination of the Tn5 barcodes and i5/i7 indices (see below). In Cusanovich et al., 2015 and Cusanovich et al., 2018, they refer the exact method to an early study that was published in Nature Genetics (Amini et al., 2014). The sequences described here are taken from their Supplementary Table 4 from the Nature Genetics paper and the Supplementary Table 12 from the Nature 2018 paper. Note the Tn5 sequences here are different from the Illumina Nextera Kit.


Adapter and primer sequences:

Barcoded Tn5 sequence s5: 5'- TCGTCGGCAGCGTCTCCACGC[8-bp Tn5 index]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG -3'

Barcoded Tn5 sequence s7: 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[8-bp Tn5 index]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG -3'

Tn5 binding site 19-bp Mosaic End (ME) bottom: 5'- /Phos/CTGTCTCTTATACACATCT -3'

P5 index primer entry point (s5): 5'- TCGTCGGCAGCGTCTCCACGC -3'

P7 index primer entry point (s7): 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC -3'

P5 index primer: 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCTCCACGC -3'

P7 index primer: 5'- CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGGCTGTCCCTGTCC -3'

Read 1 sequencing primer: 5'- GCGATCGAGGACGGCAGATGTGTATAAGAGACAG -3'

Index 1 sequencing primer (i7): 5'- CTGTCTCTTATACACATCTGAGGCGGAGACGGTG -3'

Index 2 sequencing primer (i5): 5'- CTGTCTCTTATACACATCTGCCGTCCTCGATCGC -3'

Read 2 seuquencing primer: 5'- CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG -3'

Step-by-step library generation

(1) Anneal Barcoded Tn5 sequences s5/s7 and Tn5 binding site 19-bp Mosaic End (ME) bottom strand to assemble Tn5 transposome:

Tn5 dimer

(2) Sort limited nuclei into wells, and perform tagmentation using barcoded Tn5 transposome:


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

5'- TCGTCGGCAGCGTCTCCACGC[8-bp Tn5 index]GCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                                        TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[8-bp Tn5 index]CGCACCTCTGCGACGGCTGCT -5'


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

5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[8-bp Tn5 index]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                                              TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[8-bp Tn5 index]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'


Product 3 (different ends, amplifiable):

5'- TCGTCGGCAGCGTCTCCACGC[8-bp Tn5 index]GCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                                        TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[8-bp Tn5 index]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'

(3) Pool all wells, and re-distribute into wells in a new plate, and perform library ampification using indexed P5/P7 primers:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTCGTCGGCAGCGTCTCCACGC------>
                                       5'- TCGTCGGCAGCGTCTCCACGC[8-bp Tn5 index]GCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                                                                               TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[8-bp Tn5 index]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'
                                                                                                                                                                                        <------CCTGTCCCTGTCGGCTCGGGTGCTCTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(4) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTCGTCGGCAGCGTCTCCACGCNNNNNNNNGCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXX...XXXXXXCTGTCTCTTATACACATCTGAGGCGGAGACGGTGNNNNNNNNGGACAGGGACAGCCGAGCCCACGAGACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNAGCAGCCGTCGCAGAGGTGCGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXX...XXXXXXGACAGAGAATATGTGTAGACTCCGCCTCTGCCACNNNNNNNNCCTGTCCCTGTCGGCTCGGGTGCTCTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
            Illumina P5              i5            s5             8 bp                          ME             gDNA               ME                         8 bp                s7               i7          Illumina P7
                                                              Tn5 barcode                                                                                Tn5 barcode

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, these are the gDNA reads):


                                                                    5'- GCGATCGAGGACGGCAGATGTGTATAAGAGACAG------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNAGCAGCCGTCGCAGAGGTGCGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXX...XXXXXXGACAGAGAATATGTGTAGACTCCGCCTCTGCCACNNNNNNNNCCTGTCCCTGTCGGCTCGGGTGCTCTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Index 1 sequencing primer to sequence the Tn5 barcode and i7 index (bottom strand as template, 45 cycles, the first 8 bp are Tn5 barcodes, and the last 10 bp are i7 indices, the middle 27 are dark cycles):


                                                                                                                     5'- CTGTCTCTTATACACATCTGAGGCGGAGACGGTG--------ooooooooooooooooooooooooooo--------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNAGCAGCCGTCGCAGAGGTGCGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXX...XXXXXXGACAGAGAATATGTGTAGACTCCGCCTCTGCCACNNNNNNNNCCTGTCCCTGTCGGCTCGGGTGCTCTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add Index 2 sequencing primer to sequence the second index (i5 index) (top strand as template, 39 cycles, the first 8 bp are Tn5 barcodes, and the last 10 bp are i5 indices, the middle 21 are dark cycles):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTCGTCGGCAGCGTCTCCACGCNNNNNNNNGCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXX...XXXXXXCTGTCTCTTATACACATCTGAGGCGGAGACGGTGNNNNNNNNGGACAGGGACAGCCGAGCCCACGAGACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                 <---------ooooooooooooooooooooo--------CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC -5'

(4) Add Read 2 sequencing primer to sequence the second read (top strand as template, these are the gDNA reads):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTCGTCGGCAGCGTCTCCACGCNNNNNNNNGCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXX...XXXXXXCTGTCTCTTATACACATCTGAGGCGGAGACGGTGNNNNNNNNGGACAGGGACAGCCGAGCCCACGAGACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                           <-------------GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC -5'

sci-ATAC-seq3

The sci-ATAC-seq3 uses the combinatorial indexing to identify single cells without single cell isolation, which uses the same idea as sci-ATAC-seq. The difference is that sci-ATAC-seq3 achieved the combinatorial indexing via split-pool ligation. In this way, it avoids using indexed Tn5 which is difficult to produce in many labs. Therefore, regular Tn5 that used in a typical ATAC-seq experiment can be used. Cells can be identified by the unique combination of the ligated barcodes and final library PCR index as usual (see below). The sequences described here are taken from their Supplementary Table 7 from the Science paper. The procedures described here are based on their Protocols.io page.


Adapter and primer sequences:

3LV2_N5_Splint: 5'- GCCGACGACTGATTA/3ddC/ -3'

3LV2_N7_Splint: 5'- CACGAGACGACAAGT/3ddC/ -3'

3LV2_N5_Oligo_{A01..H12}_Plate{1..4} (96 x 4 = 384 oligos): 5'- CACCGCACGAGAGGT[10-bp N5 barcode]GTAATCAG -3'

3LV2_N7_Oligo_{A01..H12}_Plate{1..4} (96 x 4 = 384 oligos): 5'- CAGCACGGCGAGACT[10-bp N7 barcode]GACTTGTC -3'

3LV2_P5_PCR_{A01..H12} (96 oligos): 5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp i5]CACCGCACGAGAGGT -3'

3LV2_P7_PCR_{A01..H12} (96 oligos): 5'- CAAGCAGAAGACGGCATACGAGAT[10-bp i7]CAGCACGGCGAGACT -3'

Tn5 binding site 19-bp Mosaic End (ME) bottom: 5'- /Phos/CTGTCTCTTATACACATCT -3'

Read 1 sequencing primer: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'

Index 1 sequencing primer: 5'- CTCCGAGCCCACGAGACGACAAGTC -3'

Index 2 sequencing priemr: 5'- ACACATCTGACGCTGCCGACGACTGATTAC -3'

Read 2 sequencing primer: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'

Step-by-step library generation

(1) Tagmentation on bulk nuclei in each individual wells (50k nuclei per well) using regular Tn5 from Illumina (will create 9-bp gap):

Tn5 dimer 

Product 1 (s5 at both ends):

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                  TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'


Product 2 (s7 at both ends):

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


Product 3 (different ends):

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

(2) Use T4 PNK to add phosphate to the 5' of the tagged fragments:


Product 1:

5'- pTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                   TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCTp -5'


Product 2:

5'- pGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                    TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGp -5'


Product 3:

5'- pTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                   TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGp -5'

(3) Add 3LV2_N5_Splint and 3LV2_N5_Oligo_{A01..H12}_Plate{1..4} oligos for N5 barcode ligation:


Product 1:

5'- CACCGCACGAGAGGT[10-bp N5 barcode]GTAATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT      GCCGACGACTGATTAC -3'
                                 3'- CATTAGTCAGCAGCCG      TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCTGACTAATG[10-bp N5 barcode]TGGAGAGCACGCCAC -5'


Product 2 (unable to ligate, omitted afterwards):

5'- pGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                    TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGp -5'


Product 3:

5'- CACCGCACGAGAGGT[10-bp N5 barcode]GTAATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                 3'- CATTAGTCAGCAGCCG      TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGp -5'

(4) Pool and redistribute to new plates with 3LV2_N7_Splint and 3LV2_N7_Oligo_{A01..H12}_Plate{1..4} oligos for N7 barcode ligation:


Product 1 (unable to ligate, omitted afterwards):

5'- CACCGCACGAGAGGT[10-bp N5 barcode]GTAATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT      GCCGACGACTGATTAC -3'
                                 3'- CATTAGTCAGCAGCCG      TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCTGACTAATG[10-bp N5 barcode]TGGAGAGCACGCCAC -5'


Product 2 (the only amplifiable fragment):

5'- CACCGCACGAGAGGT[10-bp N5 barcode]GTAATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT       CACGAGACGACAAGTC -3'
                                 3'- CATTAGTCAGCAGCCG      TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGCTGTTCAG[10-bp N7 barcode]TCAGAGCGGCACGAC -5'

(5) Pool again and redistribute to new plates, reverse crosslink and use 3LV2_P5/7_PCR_{A01..H12} for final i5/i7 index addition and library amplification:


 Gap fill-in (72C 5mins):

5'- CACCGCACGAGAGGT[10-bp N5 barcode]GTAATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACGACAAGTC[10-bp N7 barcode]AGTCTCGCCGTGCTG -3'
3'- GTGGCGTGCTCTCCA[10-bp N5 barcode]CATTAGTCAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGCTGTTCAG[10-bp N7 barcode]TCAGAGCGGCACGAC -5'


 Amplification: 

5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp i5]CACCGCACGAGAGGT---------->
                                       5'- CACCGCACGAGAGGT[10-bp N5 barcode]GTAATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACGACAAGTC[10-bp N7 barcode]AGTCTCGCCGTGCTG -3'
                                       3'- GTGGCGTGCTCTCCA[10-bp N5 barcode]CATTAGTCAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGCTGTTCAG[10-bp N7 barcode]TCAGAGCGGCACGAC -5'
                                                                                                                                                                              <-----------TCAGAGCGGCACGAC[10-bp i7]TAGAGCATACGGCAGAAGACGAAC -5'

(6) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNCACCGCACGAGAGGTNNNNNNNNNNGTAATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACGACAAGTCNNNNNNNNNNAGTCTCGCCGTGCTGNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNGTGGCGTGCTCTCCANNNNNNNNNNCATTAGTCAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGCTGTTCAGNNNNNNNNNNTCAGAGCGGCACGACNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
             Illumina P5          10-bp i5                  10 bp                 s5               ME           gDNA          ME                s7                10 bp                   10-bp i7       Illumina P7
                                                          N5 barcode                                                                                            N7 barcode

Library sequencing:

(1) Add Read 1 sequencing primer to sequence the first read (bottom strand as template, 50 cycles, these are the gDNA reads):


                                                                        5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG------>
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNGTGGCGTGCTCTCCANNNNNNNNNNCATTAGTCAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGCTGTTCAGNNNNNNNNNNTCAGAGCGGCACGACNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Index 1 sequencing primer to sequence the N7 barcode and i7 index (bottom strand as template, 35 cycles, the first 10 bp are N7 barcodes, and the last 10 bp are i7 indices, the middle 15 are dark cycles):


                                                                                                                                   5'- CTCCGAGCCCACGAGACGACAAGTC----------ooooooooooooooo--------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNGTGGCGTGCTCTCCANNNNNNNNNNCATTAGTCAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGCTGTTCAGNNNNNNNNNNTCAGAGCGGCACGACNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add Index 2 sequencing primer to sequence the N5 barcode and i5 index (top strand as template, 35 cycles, the first 10 bp are N5 barcodes, and the last 10 bp are i5 indices, the middle 15 are dark cycles):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNCACCGCACGAGAGGTNNNNNNNNNNGTAATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACGACAAGTCNNNNNNNNNNAGTCTCGCCGTGCTGNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                 <---------ooooooooooooooo----------CATTAGTCAGCAGCCGTCGCAGTCTACACA -5'

(4) Add Read 2 sequencing primer to sequence the second read (top strand as template, 50 cycles, these are the gDNA reads):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNCACCGCACGAGAGGTNNNNNNNNNNGTAATCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACGACAAGTCNNNNNNNNNNAGTCTCGCCGTGCTGNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                              <-------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'