HyDrop-ATAC

The HyDrop protocols were published in eLife on Feb 23th, 2022 (De Rop et al. eLife 11:e73971). The authors developed an open source droplet system, HyDrop, as a hybrid method between inDrop and Drop-Seq. They optimised various steps during the library construction procedures and utilised a dissolvable hydrogel beads to improve barcoded primer release and diffusion.

Both scRNA-seq and scATAC-seq can be performed on the HyDrop platform. In this page, Hydrop-ATAC is presented. This page is basically a recreation of the Supplementary Files 1 & 2 from the publication. Detailed protocol can be found here: protocols.io.

For scRNA-seq on the HyDrop platform, go to the HyDrop-RNA page for a detailed step-by-step illustration.

Adapter and primer sequences:

* The complete full list of oligo sequences can be found in this spreadsheet from their protocols.io page: 20210712_supp_methods_table_hydrop_oligonucleotide_list.xlsx.

Sequence used during the experiment:

Barcoded beads-oligo: |--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC[10-bp barcode2]CGAGTACCCT[10-bp barcode3]GTCTCGTGGGCTCGG -3'

HYi7: 5'- CAAGCAGAAGACGGCATACGAGAT[10-bp index]CTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC - 3'

HYi5: 5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp index]TCGTCGGCAGCGTCAGATGTG - 3'

Nextera Index 1 sequencing primer: 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'

Nextera Index 2 sequencing primer: 5'- CTGTCTCTTATACACATCTGACGCTGCCGACGA -3'

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

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

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Sequence used for barcoded bead generation (before the experiment):

Acrydite_primer: 5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC -3'

20200130_plate-1-96: 5' - GCAGTAGCTG[10-bp barcode1]GTACTCTGCG - 3'

20200130_plate-2-96: 5' - AGGGTACTCG[10-bp barcode2]GCAGTAGCTG - 3'

20200130_plate-3-96-ATACseq: 5'- CCGAGCCCACGAGAC[10-bp barcode3]AGGGTACTCG -3'

* The following oligos are only used for QC of the beads, not really used in the real experiments, so they are not drawn in the workflow in this page:

Anti-Acrydite_FAM: 5'- /56-FAM/TTTTTGTACTCTGCGTTGATACCAC -3'

Anti-ATAC_FAM: 5'- /56-FAM/AAAAAACCGAGCCCACGAGAC -3'

Anti-BC1_FAM: 5'- /56-FAM/TTTTTCTATCCGTCAGTAC -3'

Anti-BC2_FAM: 5'- /56-FAM/TTTTTACACGTTGTGGCAG -3'

Anti-BC3_FAM: 5'- /56-FAM/TTTTTCTCCTATCATAGGG -3'

Step-by-step generation of barcoded beads:

(1) Form dissolvable acrylimide gel beads together with Acrydite_primer:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC -3'

(2) Split the gel beads into wells in 20200130_plate-1-96, and perform extension:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC---->
                                                                <----GCGTCTCATG[10-bp barcode1]GTCGATGACG -5'

(3) This is the product after the first extension:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC -3'
                       3'- AAAAAAAATTATGCTGAGTGATATCCCTTCGTCACCATAGTTGCGTCTCATG[10-bp barcode1]GTCGATGACG -5'

(4) Pooling, denature by NaOH, and get rid of top strand:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC -3'

(5) Split again into wells in 20200130_plate-2-96, and perform extension:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC---->
                                                                                          <----GTCGATGACG[10-bp barcode2]GCTCATGGGA -5'

(6) This is the product of the second extension:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC[10-bp barcode2]CGAGTACCCT -3'
                       3'- AAAAAAAATTATGCTGAGTGATATCCCTTCGTCACCATAGTTGCGTCTCATG[10-bp barcode1]GTCGATGACG[10-bp barcode2]GCTCATGGGA -5'

(7) Pool again, denature by NaOH, get rid of top strand:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC[10-bp barcode2]CGAGTACCCT -3'

(8) Split again into wells in 20200130_plate-3-96-ATACseq, and perform extension:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC[10-bp barcode2]CGAGTACCCT---->
                                                                                                                    <----GCTCATGGGA[10-bp barcode3]CAGAGCACCCGAGCC -5'

(9) This is the product of the third extension:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC[10-bp barcode2]CGAGTACCCT[10-bp barcode3]GTCTCGTGGGCTCGG -3'
                       3'- AAAAAAAATTATGCTGAGTGATATCCCTTCGTCACCATAGTTGCGTCTCATG[10-bp barcode1]GTCGATGACG[10-bp barcode2]GCTCATGGGA[10-bp barcode3]CAGAGCACCCGAGCC -5'

(10) Pool again, denature by NaOH, get rid of the bottom strand, neutralise, wash and ready to use (96 x 96 x 96 different combination of barcodes 1, 2 & 3):


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC[10-bp barcode2]CGAGTACCCT[10-bp barcode3]GTCTCGTGGGCTCGG -3'

Step-by-step library generation

(1) Bulk Tn5 tagging by incubation of nuclei and Tn5:

(2) There are 3 different products after step (1) (will create 9 bp gap):


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

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


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

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


Product 3 (different ends, amplifiable):

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

(3) Droplet capture and subquent gap fill-in (3.1) and linear PCR to add cell barcode (3.2):

(3.1) Fill-in (the first step of linear PCR, 72 degree):


5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX------------------------------------------>
    <-----------------------------------------XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(3.2) Linear PCR to add cell barcodes:


5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC
    AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                  <----------------GGCTCGGGTGCTCTG[10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAAGGGATATCACTCAGCATAATTTTTTTT -5'--|

(4) Product recovered from the droplets:


5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC[10-bp barcode3]AGGGTACTCG[10-bp barcode2]GCAGTAGCTG[10-bp barcode1]GTACTCTGCGTTGATACCACTGCTTCCCTATAGTGAGTCGTATTAAAAAAAA -3'
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAAGGGATATCACTCAGCATAATTTTTTTT -5'

(5) PCR amplification with index primers HYi7 and HYi5 to get the library:


5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp index]TCGTCGGCAGCGTCAGATGTG-------------------->
                                          5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC[10-bp barcode3]AGGGTACTCG[10-bp barcode2]GCAGTAGCTG[10-bp barcode1]GTACTCTGCGTTGATACCACTGCTTCCCTATAGTGAGTCGTATTAAAAAAAA -3'
                                          3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAAGGGATATCACTCAGCATAATTTTTTTT -5'
                                                                                                                                                                                           <--------------------CATGAGACGCAACTATGGTGACGAAGG
                                                                                                                                                                                                                                           CGCCTGTC[10-bp index]TAGAGCATACGGCAGAAGACGAAC -5'

(6) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNNNAGGGTACTCGNNNNNNNNNNGCAGTAGCTGNNNNNNNNNNGTACTCTGCGTTGATACCACTGCTTCCGCGGACAGNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNNNTCCCATGAGCNNNNNNNNNNCGTCATCGACNNNNNNNNNNCATGAGACGCAACTATGGTGACGAAGGCGCCTGTCNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
            Illumina P5            10-bp         s5              ME           gDNA           ME               s7          10-bp               10-bp               10-bp                                       10-bp          Illumina P7
                              sample index                                                                              barcode3            barcode2            barcode1                                  sample index
                                                                                                                                                                                                  (deprecated, not sequenced)

Library sequencing using Illumina primers

(1) Nextera Read 1 sequencing primer is used to sequence the first read (bottom strand as template, 50 cycles, open chromatin read):


                                       5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNNNTCCCATGAGCNNNNNNNNNNCGTCATCGACNNNNNNNNNNCATGAGACGCAACTATGGTGACGAAGGCGCCTGTCNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Nextera Index 1 sequencing primer is used to sequence the cell barcodes (bottom strand as template, 52 cycles):


                                                                                 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC--------------------------------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNNNTCCCATGAGCNNNNNNNNNNCGTCATCGACNNNNNNNNNNCATGAGACGCAACTATGGTGACGAAGGCGCCTGTCNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Nextera Index 2 sequencing primer is used to sequence the sample index (top strand as template, 10 cycles):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNNNAGGGTACTCGNNNNNNNNNNGCAGTAGCTGNNNNNNNNNNGTACTCTGCGTTGATACCACTGCTTCCGCGGACAGNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                 <---------AGCAGCCGTCGCAGTCTACACATATTCTCTGTC -5'

(4) Nextera Read 2 sequencing primer to sequence the second read (top strand as template, 50 cycles, open chromatin read):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNNNAGGGTACTCGNNNNNNNNNNGCAGTAGCTGNNNNNNNNNNGTACTCTGCGTTGATACCACTGCTTCCGCGGACAGNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                            <--------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'