# Drop-seq / Seq-Well

In the original pulication in Cell 161, 1202-1214 (2015), there are two batches of beads, with only two base pairs difference. Here, Beads-oligo-dT-seqA was used as demonstration. Seq-Well used exact the same oligo design with Drop-seq with Beads-oligo-dT-seqB, which was published in Nature Methods 14, 395–398 (2017).

## Adapter and primer sequences:

Beads-oligo-dT-seqA: |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT[12-bp cell barcode][8-bp UMI]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT -3'

Beads-oligo-dT-seqB: |--5'- TTTTTTTAAGCAGTGGTATCAACGCAGAGTAC[12-bp cell barcode][8-bp UMI]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT -3'

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

ISPCR: 5′- AAGCAGTGGTATCAACGCAGAGT -3′

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

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

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

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Library PCR primer 1: 5'- AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC -3'

Library PCR primer 2 (this is basically Nextera N7xx): 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp i7 index]GTCTCGTGGGCTCGG -3'

Read 1 sequencing primer (seqA): 5'- GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACGT -3'

Read 1 sequencing primer (seqB): 5'- GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC -3'

Read 2 sequencing primer: 5'- GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG -3'

i7 index sequencing primer: 5'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC -3'

## Step-by-step library generation

### (1) mRNA capture using Beads-oligo-dT in the droplets:

    5'- XXXXXXXXXXXXXXXXXXXB(A)n
                     <----NV(T)30[8-bp UMI][12-bp cell barcode]TGCATGAGACGCAACTATGGTGACGAATTTTTTTT -5'--|

### (2) Break droplets (i.e. droplets are merely used as a cell capture chamber), reverse transcription for all cells in one reaction, the terminal tranferase acitivity of MMLV adds extra Cs:

    5'- XXXXXXXXXXXXXXXXXXXB(A)n
     CCCXXXXXXXXXXXXXXXXXXNV(T)30[8-bp UMI][12-bp cell barcode]TGCATGAGACGCAACTATGGTGACGAATTTTTTTT -5'--|

### (3) Adding TSO for second strand synthesis:

    5'- AAGCAGTGGTATCAACGCAGAGTGAATGGGXXXXXXXXXXXXXXXXXXXX(A)n---->
                            <------CCCXXXXXXXXXXXXXXXXXXXX(T)30[8-bp UMI][12-bp cell barcode]TGCATGAGACGCAACTATGGTGACGAATTTTTTTT -5'--|

### (4) Adding ISPCR for single primer cDNA amplification:( i.e. semi-suppressive PCR )

    5'- AAGCAGTGGTATCAACGCAGAGT---->
    5'- AAGCAGTGGTATCAACGCAGAGTGAATGGGXXXXX...XXXXX(pA)[8-bp UMI][12-bp cell barcode]ACGTACTCTGCGTTGATACCACTGCTTAAAAAAAA
        TTCGTCACCATAGTTGCGTCTCACTTACCCXXXXX...XXXXX(dT)[8-bp UMI][12-bp cell barcode]TGCATGAGACGCAACTATGGTGACGAATTTTTTTT -5'--|
                                                                                    <----TGAGACGCAACTATGGTGACGAA -5'

### (5) Nextera tagmentation on amplified cDNA (will create 9-bp gap):

    Product 1 (5'-end of cDNA, with s5 sequence, not amplifiable due to the use of Nextera N7xx for library amplification, see step 6):

    5'- AAGCAGTGGTATCAACGCAGAGTGAATGGGXXX...XXX         CTGTCTCTTATACACATCT
        TTCGTCACCATAGTTGCGTCTCACTTACCCXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'

    Product 2 (5'-end of cDNA, with s7 sequence, not amplifiable due to Library PCR Primer 1 ends with "AC" which cannot to be annealed to the template, see step 6):

    5'- AAGCAGTGGTATCAACGCAGAGTGAATGGGXXX...XXX         CTGTCTCTTATACACATCT
        TTCGTCACCATAGTTGCGTCTCACTTACCCXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

    Product 3 (3'-end of cDNA, with s5 sequence, not amplifiable due to the use of Nextera N7xx for library amplification, see step 6):

    |--5'- TTTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT[12-bp cell barcode][8-bp UMI](dT)XXX...XXX         CTGTCTCTTATACACATCT
           AAAAAAAATTCGTCACCATAGTTGCGTCTCATGCA[12-bp cell barcode][8-bp UMI](pA)XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'

    Product 4 (3'-end of cDNA, with s7 sequence, the only amplifiable fragment which will be used for library prep and sequencing, see step 6):

    |--5'- TTTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT[12-bp cell barcode][8-bp UMI](dT)XXX...XXX         CTGTCTCTTATACACATCT
           AAAAAAAATTCGTCACCATAGTTGCGTCTCATGCA[12-bp cell barcode][8-bp UMI](pA)XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

### (6) Add Library PCR Primer 1 & 2 to amplify library:

    5'- AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC-------->
                                  |--5'- TTTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT[12-bp cell barcode][8-bp UMI](dT)XXX...XXX         CTGTCTCTTATACACATCT
                                         AAAAAAAATTCGTCACCATAGTTGCGTCTCATGCA[12-bp cell barcode][8-bp UMI](pA)XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                                                          <--------GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'

### (7) Final library structure:

    5'- AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACGTNNNNNNNNNNNNNNNNNNNN(dT)XXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
        TTACTATGCCGCTGGTGGCTCTAGATGTGCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCATGCANNNNNNNNNNNNNNNNNNNN(pA)XXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
                  Illumina P5                           ISPCR/TSO            12bp cell     8bp        cDNA           ME             s7            i7          Illumina P7
                                                                              barcode      UMI

## Library sequencing:

### (1) Add Read 1 sequencing primer (seqA) to sequence the first read (bottom strand as template, sequence 12-bp cell barcode and 8-bp UMI):

                                 5'- GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACGT------------------->
    3'- TTACTATGCCGCTGGTGGCTCTAGATGTGCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCATGCANNNNNNNNNNNNNNNNNNNN(pA)XXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

### (2) Add i7 index sequencing primer to sequence the i7 index (bottom strand as template):

                                                                                                         5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC------->
    3'- TTACTATGCCGCTGGTGGCTCTAGATGTGCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCATGCANNNNNNNNNNNNNNNNNNNN(pA)XXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

### (3) Cluster regeneration, add Read 2 sequencing primer to sequence the second read (top strand as template, sequence cDNA):

    5'- AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACGTNNNNNNNNNNNNNNNNNNNN(dT)XXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                     <-------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'