HyDrop-RNA

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-RNA is presented. This page is basically a recreation of the Supplementary Files 1 & 3 from the publication. Detailed protocol can be found here: protocols.io.

For scATAC-seq on the HyDrop platform, go to the HyDrop-ATAC 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][8-bp UMI]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT -3'

Template Switching Oligo (TSO): 5'- AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG -3'

TSO-P: 5'- AAGCAGTGGTATCAACGCAGAGT -3'

NEB Hairpin adapter:

    
            ACACTCTTTCCCTACACGAC
            |                   GCTCTTCCGATCT -3'
            U                   CGAGAAGGCTAG -5'
            \CTGACCTCAAGTCTGCACA

HYi5_TruSeq: 5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp index]ACACTCTTTCCCTACACGACGCT -3'

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

TruSeq Read 1: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT -3'

HyDrop_CustSeq_Short: 5'- GTACTCTGCGTTGATACCACTGCTTCCGCGGACAG -3'

HyDrop_CustSeq_R2: 5'- CTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC -3'

Index 2 Sequencing Primer: 5'- AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT -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-RNAseq: 5'- AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA[8-bp UMI][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-RNA_FAM: 5'- /56-FAM/AAAAAAAAAAAAAAAAAAAA -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-RNAseq, and perform extension:


|--5'- /5Acryd//iThioMC6-D/TTTTTTTTAATACGACTCACTATAGGGAAGCAGTGGTATCAACGCAGAGTAC[10-bp barcode1]CAGCTACTGC[10-bp barcode2]CGAGTACCCT---->
                                                                                                                    <----GCTCATGGGA[10-bp barcode3][8-bp UMI]AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA -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][8-bp UMI](T)30 -3'
                       3'- AAAAAAAATTATGCTGAGTGATATCCCTTCGTCACCATAGTTGCGTCTCATG[10-bp barcode1]GTCGATGACG[10-bp barcode2]GCTCATGGGA[10-bp barcode3][8-bp UMI](A)30 -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][8-bp UMI](T)30 -3'

Step-by-step library generation

(1) Lysis, mRNA anneals to poly-T barcodes on the beads:


5'- XXX...XXXAAAAAAAAAAAAAAA...AAAAAAAAAAAAAAA
        <------TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[8-bp UMI][10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAAGGGATATCACTCAGCATAATTTTTTTT -5'--|

(2) The terminal transferase activity of MMLV adds extra Cs:


    5'- XXX...XXX(pA)
     CCCXXX...XXX(dT)[8-bp UMI][10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAAGGGATATCACTCAGCATAATTTTTTTT -5'--|

(3) Perform template switching with TSO:


5'- AAGCAGTGGTATCAACGCAGAGTGAATGGGXXX...XXX(pA)
                  <------------CCCXXX...XXX(dT)[8-bp UMI][10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAAGGGATATCACTCAGCATAATTTTTTTT -5'--|

(4) Break emulsion, purify cDNA:


3'- TTCGTCACCATAGTTGCGTCTCACTTACCCXXX...XXX(dT)[8-bp UMI][10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAAGGGATATCACTCAGCATAATTTTTTTT -5'

(5) Use the TSO-P for single primer cDNA amplification:( i.e. semi-suppressive PCR ):


5'- AAGCAGTGGTATCAACGCAGAGT--------->
3'- TTCGTCACCATAGTTGCGTCTCACTTACCCXXX...XXX(dT)[8-bp UMI][10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAAGGGATATCACTCAGCATAATTTTTTTT -5'
                                                                                                                     <---------TGAGACGCAACTATGGTGACGAA -5'

(5) Purify amplified cDNA:


5'- AAGCAGTGGTATCAACGCAGAGTGAATGGGXXX...XXX(pA)[8-bp UMI][10-bp barcode3]AGGGTACTCG[10-bp barcode2]GCAGTAGCTG[10-bp barcode1]GTACTCTGCGTTGATACCACTGCTT -3'
3'- TTCGTCACCATAGTTGCGTCTCACTTACCCXXX...XXX(dT)[8-bp UMI][10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAA -5'

(6) Fragmentation and dA-tailing (three different products):


Product 1 (the 5' end of the gene):

5'-  AAGCAGTGGTATCAACGCAGAGTGAATGGGXXX...XXXA -3'
3'- ATTCGTCACCATAGTTGCGTCTCACTTACCCXXX...XXX  -5'



Product 2 (the middle part of the gene):

5'-  XXXXXXXXXX...XXXXXXXXXXA -3'
3'- AXXXXXXXXXX...XXXXXXXXXX  -5'



Product 3 (the 3' end of the gene):

5'-  XXX...XXX(pA)[8-bp UMI][10-bp barcode3]AGGGTACTCG[10-bp barcode2]GCAGTAGCTG[10-bp barcode1]GTACTCTGCGTTGATACCACTGCTTA -3'
3'- AXXX...XXX(dT)[8-bp UMI][10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAA  -5'

(7) NEB Hairpin adapter ligation:


Product 1 (the 5' end of the gene):

ACACTCTTTCCCTACACGAC                                                                 ACACGTCTGAACTCCAGTC\
|                   GCTCTTCCGATCTAAGCAGTGGTATCAACGCAGAGTGAATGGGXXX...XXXAGATCGGAAGAGC                   U
U                   CGAGAAGGCTAGATTCGTCACCATAGTTGCGTCTCACTTACCCXXX...XXXTCTAGCCTTCTCG                   |
\CTGACCTCAAGTCTGCACA                                                                 CAGCACATCCCTTTCTCACA



Product 2 (the middle part of the gene):

ACACTCTTTCCCTACACGAC                                   ACACGTCTGAACTCCAGTC\
|                   GCTCTTCCGATCTXXX...XXXAGATCGGAAGAGC                   U
U                   CGAGAAGGCTAGAXXX...XXXTCTAGCCTTCTCG                   |
\CTGACCTCAAGTCTGCACA                                   CAGCACATCCCTTTCTCACA



Product 3 (the 3' end of the gene):

ACACTCTTTCCCTACACGAC                                                                                                                                              ACACGTCTGAACTCCAGTC\
|                   GCTCTTCCGATCTXXX...XXX(pA)[8-bp UMI][10-bp barcode3]AGGGTACTCG[10-bp barcode2]GCAGTAGCTG[10-bp barcode1]GTACTCTGCGTTGATACCACTGCTTAGATCGGAAGAGC                   U
U                   CGAGAAGGCTAGAXXX...XXX(dT)[8-bp UMI][10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAATCTAGCCTTCTCG                   |
\CTGACCTCAAGTCTGCACA                                                                                                                                              CAGCACATCCCTTTCTCACA

(8) NEB USER Enzyme digestion:


Product 1 (the 5' end of the gene, not amplifiable due to the HYi7 PCR primer used in the next step ends with AC):

5'- ACACTCTTTCCCTACACGAC                                                                 ACACGTCTGAACTCCAGTC -3'
                        GCTCTTCCGATCTAAGCAGTGGTATCAACGCAGAGTGAATGGGXXX...XXXAGATCGGAAGAGC
                        CGAGAAGGCTAGATTCGTCACCATAGTTGCGTCTCACTTACCCXXX...XXXTCTAGCCTTCTCG
 3'- CTGACCTCAAGTCTGCACA                                                                 CAGCACATCCCTTTCTCACA -5'



Product 2 (the middle part of the gene, not amplifiable due to the primers used in the next step):

5'- ACACTCTTTCCCTACACGAC                                   ACACGTCTGAACTCCAGTC -3'
                        GCTCTTCCGATCTXXX...XXXAGATCGGAAGAGC
                        CGAGAAGGCTAGAXXX...XXXTCTAGCCTTCTCG
 3'- CTGACCTCAAGTCTGCACA                                   CAGCACATCCCTTTCTCACA -5'



Product 3 (the 3' end of the gene, the only amplifiable fragment):

5'- ACACTCTTTCCCTACACGAC                                                                                                                                              ACACGTCTGAACTCCAGTC -3'
                        GCTCTTCCGATCTXXX...XXX(pA)[8-bp UMI][10-bp barcode3]AGGGTACTCG[10-bp barcode2]GCAGTAGCTG[10-bp barcode1]GTACTCTGCGTTGATACCACTGCTTAGATCGGAAGAGC
                        CGAGAAGGCTAGAXXX...XXX(dT)[8-bp UMI][10-bp barcode3]TCCCATGAGC[10-bp barcode2]CGTCATCGAC[10-bp barcode1]CATGAGACGCAACTATGGTGACGAATCTAGCCTTCTCG
 3'- CTGACCTCAAGTCTGCACA                                                                                                                                              CAGCACATCCCTTTCTCACA -5'

(9) Add HYi5_TruSeq and HYi7 primers to perform PCR to amplify the library:


Note that only the top strand of Product 3 can be amplified by both primers exponentially:


5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp index]ACACTCTTTCCCTACACGACGCT--------->
                                          5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCTXXX...XXX(pA)[8-bp UMI][10-bp barcode3]AGGGTACTCG[10-bp barcode2]GCAGTAGCTG[10-bp barcode1]GTACTCTGCGTTGATACCACTGCTTAGATCGGAAGAGCACACGTCTGAACTCCAGTC -3'
                                                                                                                                                     <--------------------CATGAGACGCAACTATGGTGACGAA
                                                                                                                                                                                                   GGCGCCTGTC[10-bp index]TAGAGCATACGGCAGAAGACGAAC -5'

(6) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTXXX...XXX(pA)NNNNNNNNNNNNNNNNNNAGGGTACTCGNNNNNNNNNNGCAGTAGCTGNNNNNNNNNNGTACTCTGCGTTGATACCACTGCTTCCGCGGACAGNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAXXX...XXX(dT)NNNNNNNNNNNNNNNNNNTCCCATGAGCNNNNNNNNNNCGTCATCGACNNNNNNNNNNCATGAGACGCAACTATGGTGACGAAGGCGCCTGTCNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
            Illumina P5            10-bp            TruSeq Read 1              cDNA        8-bp     10-bp               10-bp               10-bp              TSO                       10-bp         Illumina P7
                               sample index                                                UMI     barcode3            barcode2            barcode1                                  sample index

Library sequencing using Illumina primers

(1) Add TruSeq Read 1 sequencing primer to sequence the first read (bottom strand as template, cDNA reads, >50 cycles):


                                       5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAXXX...XXX(dT)NNNNNNNNNNNNNNNNNNTCCCATGAGCNNNNNNNNNNCGTCATCGACNNNNNNNNNNCATGAGACGCAACTATGGTGACGAAGGCGCCTGTCNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add HyDrop_CustSeq_Short primer to sequence the sample index at the i7 side (bottom strand as template, 10 cycles):


                                                                                                                                               5'- GTACTCTGCGTTGATACCACTGCTTCCGCGGACAG--------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAXXX...XXX(dT)NNNNNNNNNNNNNNNNNNTCCCATGAGCNNNNNNNNNNCGTCATCGACNNNNNNNNNNCATGAGACGCAACTATGGTGACGAAGGCGCCTGTCNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Add Index 2 sequencing primer to sequence the sample index at the i5 side (top strand as template, 10 cycles):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTXXX...XXX(pA)NNNNNNNNNNNNNNNNNNAGGGTACTCGNNNNNNNNNNGCAGTAGCTGNNNNNNNNNNGTACTCTGCGTTGATACCACTGCTTCCGCGGACAGNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                 <---------TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -5'

(4) Add HyDrop_CustSeq_R2 primer to sequence the second read (top strand as template, 58 cycles, these are cell barcodes and UMI):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTXXX...XXX(pA)NNNNNNNNNNNNNNNNNNAGGGTACTCGNNNNNNNNNNGCAGTAGCTGNNNNNNNNNNGTACTCTGCGTTGATACCACTGCTTCCGCGGACAGNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                         <---------------------------------------------------------CATGAGACGCAACTATGGTGACGAAGGCGCCTGTC -5'