STRT-seq-2i

STRT-seq

STRT-seq was originally pulished in Genome Res. 21: 1160-1167 (2011). One year after that, the authors published the detailed protocol in Nature Protocols 7, 813-828 (2012), which is only slightly different from the original paper. The workflow shown here is based on the protocol in Nature Protocols 7, 813-828 (2012).

Adapter and primer sequences:

oligo-dTVN: 5'-Bio- TTAAGCAGTGGTATCAACGCAGAGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN -3'

Barcoded Template Switching Oligo (TSO): 5'- AAGCAGTGGTATCAACGCAGAGTGCAGTGCT[6-bp cell barcode]rGrGrG -3'

ISPCR: 5'-Bio- AAGCAGTGGTATCAACGCAGAGT -3'

^*Solexa P1 adapter: 5'- AATGATACGGCGACCACCGA -3'

^*Solexa P2 adapter: 5'- CAAGCAGAAGACGGCATACGA -3'

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

Library PCR primer 2: 5'- CAAGCAGAAGACGGCATACGAG -3'

Doube stranded P2 adapter with T overhang:

5'- CAAGCAGAAGACGGCATACGAGCTCTTCCGATC*T -3'
     3'- GTTCGTCTTCTGCCGTATGCTCGAGAAGGCTAG  -5'

* Recent sequencing libraries use Illumina P5/P7 adapters, instead of Solexa P1/P2 adapters.

Step-by-step library generation

(1) Anneal oligo-dTVN to mRNA and reverse transcription using MMLV:


5'- XXXXXXXXXXXXXXXXXXXB(A)_n
                 <----NV(T)₃₀CAGCTGAGACGCAACTATGGTGACGAATT -Bio-5'

(2) After reverse transcription, the terminal tranferase acitivity of MMLV add extra Cs:


5'- XXXXXXXXXXXXXXXXXXXB(A)_n
 CCCXXXXXXXXXXXXXXXXXXNV(T)₃₀CAGCTGAGACGCAACTATGGTGACGAATT -Bio-5'

(3) Adding Barcoded TSO for second strand synthesis:


5'- AAGCAGTGGTATCAACGCAGAGTGCAGTGCT[6-bp cell barcode]GGGXXXXXXXXXXXXXXXXXXXX(A)_n
                                                      CCCXXXXXXXXXXXXXXXXXXXX(T)₃₀CAGCTGAGACGCAACTATGGTGACGAATT -Bio-5'

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


5'-Bio- AAGCAGTGGTATCAACGCAGAGT---->
5'-     AAGCAGTGGTATCAACGCAGAGTGCAGTGCT[6-bp cell barcode]GGGXXXXXXX...XXXXXXX(pA)GTCGACTCTGCGTTGATACCACTGCTTAA
        TTCGTCACCATAGTTGCGTCTCACGTCACGA[6-bp cell barcode]CCCXXXXXXX...XXXXXXX(dT)CAGCTGAGACGCAACTATGGTGACGAATT -Bio-5'
                                                                                 <----TGAGACGCAACTATGGTGACGAA -Bio-5'

(5) Streptavidin beads binding, and cDNA fragmentation using DNase I in the presence of Mn²⁺, which preferentially causes double-stranded breaks:


Product 1 (5'-end of cDNA):

|--5'-Bio- AAGCAGTGGTATCAACGCAGAGTGCAGTGCT[6-bp cell barcode]GGGXXXXXXX...XXXXXXX
           TTCGTCACCATAGTTGCGTCTCACGTCACGA[6-bp cell barcode]CCCXXXXXXX...XXXXXXX -5'




Product 2 (3'-end of cDNA):

|--5'-Bio- AAGCAGTGGTATCAACGCAGAGTCGAC(dT)XXXXXXXXXX...XXXXXXXXXX
           TTCGTCACCATAGTTGCGTCTCAGCTG(pA)XXXXXXXXXX...XXXXXXXXXX -5'

(6) A tailing:


Prodcut 1 (5'-end of cDNA):

|--5'-Bio- AAGCAGTGGTATCAACGCAGAGTGCAGTGCT[6-bp cell barcode]GGGXXXXXXX...XXXXXXX*A
           TTCGTCACCATAGTTGCGTCTCACGTCACGA[6-bp cell barcode]CCCXXXXXXX...XXXXXXX   -5'




Product 2 (3'-end of cDNA):

|--5'-Bio- AAGCAGTGGTATCAACGCAGAGTCGAC(dT)XXXXXXXXXX...XXXXXXXXXX*A
           TTCGTCACCATAGTTGCGTCTCAGCTG(pA)XXXXXXXXXX...XXXXXXXXXX   -5'

(7) Ligation with doube stranded P2 adapter with T overhang, and at the same time, SalI digestion (5'...GTCGAC...3'):


Product 1 (5'-end of cDNA, will not be affected by SalI):

|--5'-Bio- AAGCAGTGGTATCAACGCAGAGTGCAGTGCT[6-bp cell barcode]GGGXXXXXXX...XXXXXXX*AGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
           TTCGTCACCATAGTTGCGTCTCACGTCACGA[6-bp cell barcode]CCCXXXXXXX...XXXXXXX TCTAGCCTTCTCGAGCATACGGCAGAAGACGAAC -5'




Product 2 (3'-end of cDNA, will be cut by SalI, this product is omitted later since it cannot be amplified:):

|--5'-Bio- AAGCAGTGGTATCAACGCAGAG   TCGAC(dT)XXXXXXXXXX...XXXXXXXXXX*AGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
           TTCGTCACCATAGTTGCGTCTCAGCT   G(pA)XXXXXXXXXX...XXXXXXXXXX TCTAGCCTTCTCGAGCATACGGCAGAAGACGAAC -5'

(8) Add Library PCR Primers 1&2 for amplification:


5'- AATGATACGGCGACCACCGAGATCTAAGCAGTGGTATCAACGCAGAGT----->
                  |--5'-Bio- AAGCAGTGGTATCAACGCAGAGTGCAGTGCT[6-bp cell barcode]GGGXXXXXXX...XXXXXXXAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
                             TTCGTCACCATAGTTGCGTCTCACGTCACGA[6-bp cell barcode]CCCXXXXXXX...XXXXXXXTCTAGCCTTCTCGAGCATACGGCAGAAGACGAAC -5'
                                                                                                        <------GAGCATACGGCAGAAGACGAAC -5'

(9) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTAAGCAGTGGTATCAACGCAGAGTGCAGTGCTNNNNNNGGGXXXXXXX...XXXXXXXAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
    TTACTATGCCGCTGGTGGCTCTAGATTCGTCACCATAGTTGCGTCTCACGTCACGANNNNNNCCCXXXXXXX...XXXXXXXTCTAGCCTTCTCGAGCATACGGCAGAAGACGAAC -5'
          Solexa P1                ISPCR/TSO                  6bp                                       Solexa P2
                                                             cell
                                                            barcode

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, the first 6-bp of the read will be the cell barcode):


                    5'- GATCTAAGCAGTGGTATCAACGCAGAGTGCAGTGCT--------->
3'- TTACTATGCCGCTGGTGGCTCTAGATTCGTCACCATAGTTGCGTCTCACGTCACGANNNNNNCCCXXXXXXX...XXXXXXXTCTAGCCTTCTCGAGCATACGGCAGAAGACGAAC -5'

(2) Cluster regeneration, add read 2 sequencing primer to sequence the second read (top strand as template, not sure whether this step is actually perfromed):


5'- AATGATACGGCGACCACCGAGATCTAAGCAGTGGTATCAACGCAGAGTGCAGTGCTNNNNNNGGGXXXXXXX...XXXXXXXAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
                                                                              <-------TCTAGCCTTCTCGAGCATACGGCAGAAGACGAAC -5'

STRT-seq-C1

Adapter and primer sequences:

C1-P1-T31: 5'-Bio- AATGATACGGCGACCACCGATCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT -3'

C1-P1-RNA-TSO: 5'-Bio- AAUGAUACGGCGACCACCGAU[5-bp UMI]GGG -3'

C1-P1-PCR-2 primer: 5'-Bio- GAATGATACGGCGACCACCGAT -3'

Barcoded C1-Tn5 top (including 19-bp Mosaic End (ME)): 5'- CAAGCAGAAGACGGCATACGA[8-bp cell barcode]GCGTCAGATGTGTATAAGAGACAG -3'

Universal C1-Tn5 bottom: 5'- CTGTCTCTTATACACATCTGACGC -3'

Annealed Tn5 oligos for Tn5 transposome assembly:

5'- CAAGCAGAAGACGGCATACGA[8-bp cell barcode]GCGTCAGATGTGTATAAGAGACAG -3'
                                                  3'- TCTACACATATTCTCTGTC -5'

^*Solexa P1 adapter: 5'- AATGATACGGCGACCACCGA -3'

^*Solexa P2 adapter: 5'- CAAGCAGAAGACGGCATACGA -3'

* Recent sequencing libraries use Illumina P5/P7 adapters, instead of Solexa P1/P2 adapters.

Step-by-step library generation

(1) Anneal C1-P1-T31 to mRNA and reverse transcription using MMLV:


5'- XXXXXXXXXXXXXXXXXXXB(A)_n
                 <----NV(T)₃₁GCTAGCCACCAGCGGCATAGTAA -Bio-5'

(2) After reverse transcription, the terminal tranferase acitivity of MMLV add extra Cs:


5'- XXXXXXXXXXXXXXXXXXXB(A)_n
 CCCXXXXXXXXXXXXXXXXXXNV(T)₃₁GCTAGCCACCAGCGGCATAGTAA -Bio-5'

(3) Adding C1-P1-RNA-TSO for second strand synthesis:


5'-Bio- AAUGAUACGGCGACCACCGAU[5-bp UMI]GGGXXXXXXXXXXXXXXXXXXXX(A)_n
                                       CCCXXXXXXXXXXXXXXXXXXXX(T)₃₁GCTAGCCACCAGCGGCATAGTAA -Bio-5'

(4) cDNA amplification using single C1-P1-PCR-2 primer:( i.e. semi-suppressive PCR )


5'-Bio- GAATGATACGGCGACCACCGAT---->
5'-Bio-  AAUGAUACGGCGACCACCGAU[5-bp UMI]GGGXXXXXXXXXXXXXXXXXXXX(pA)CGATCGGTGGTCGCCGTATCATT
         TTACTATGCCGCTGGTGGCTA[5-bp UMI]CCCXXXXXXXXXXXXXXXXXXXX(dT)GCTAGCCACCAGCGGCATAGTAA -Bio-5'
                                                                <----TAGCCACCAGCGGCATAGTAAG -Bio-5'

(5) Homemade Tn5 homodimers using C1-Tn5_top/bottom tagmentation on amplified cDNA (will create 9-bp gap):


Product 1 (5'-end of cDNA):

5'-Bio- GAATGATACGGCGACCACCGAT[5-bp UMI]GGGXXXXXXX...XXX         CTGTCTCTTATACACATCT
        CTTACTATGCCGCTGGTGGCTA[5-bp UMI]CCCXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCG[8-bp cell barcode]AGCATACGGCAGAAGACGAAC -5'

Product 2 (3'-end of cDNA):

5'-Bio- GAATGATACGGCGACCACCGATCG(dT)XXXXXXX...XXX         CTGTCTCTTATACACATCT
        CTTACTATGCCGCTGGTGGCTAGC(pA)XXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCG[8-bp cell barcode]AGCATACGGCAGAAGACGAAC -5'

(6) Streptavidin beads binding. and PvuI cleavage (5'...CGATCG...3')


Product 1 (5'-end of cDNA) will not be affected:

|--5'-Bio- GAATGATACGGCGACCACCGAT[5-bp UMI]GGGXXXXXXX...XXX         CTGTCTCTTATACACATCT
           CTTACTATGCCGCTGGTGGCTA[5-bp UMI]CCCXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCG[8-bp cell barcode]AGCATACGGCAGAAGACGAAC -5'

Product 2 (3'-end of cDNA) will be cut by PvuI (this product is omitted later since it cannot be amplified):

|--5'-Bio- GAATGATACGGCGACCACCGAT   CG(dT)XXXXXXX...XXX         CTGTCTCTTATACACATCT
           CTTACTATGCCGCTGGTGGC   TAGC(pA)XXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCG[8-bp cell barcode]AGCATACGGCAGAAGACGAAC -5'

(7) NaOH denature Product 1 (5'-end of cDNA), and library amplification with P1 and P2 adapters using bottom strand as template:


5'-  AATGATACGGCGACCACCGAT-------->
3'- CTTACTATGCCGCTGGTGGCTA[5-bp UMI]CCCXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCG[8-bp cell barcode]AGCATACGGCAGAAGACGAAC -5'
                                                                                              <---------AGCATACGGCAGAAGACGAAC -5'

(8) Final library structure:


5'- AATGATACGGCGACCACCGATNNNNNGGGXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTGACGCNNNNNNNNTCGTATGCCGTCTTCTGCTTG
    TTACTATGCCGCTGGTGGCTANNNNNCCCXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGNNNNNNNNAGCATACGGCAGAAGACGAAC -5'
        Solexa P1          UMI          cDNA                   ME                 8bp        Solexa P2
                                                                                  cell
                                                                                 barcode

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, the first 5-bp of the read will be UMI):


5'- TTACTATGCCGCTGGTGGCTA-------->
3'- TTACTATGCCGCTGGTGGCTANNNNNCCCXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGNNNNNNNNAGCATACGGCAGAAGACGAAC -5'

(2) Add index sequencing primer to sequence cell barcodes (bottom strand as template):


                                                   5'- CTGTCTCTTATACACATCTGACGC------->
3'- TTACTATGCCGCTGGTGGCTANNNNNCCCXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGNNNNNNNNAGCATACGGCAGAAGACGAAC -5'

STRT-seq-2i

This method is similar to STRT-seq-C1, but with nanowell capture and different oigo design, and there probably a mistake in the sequence of DI-Read1-Seq at this time of the preprint (29-08-2017), where there should be only five Ns in DI-Read1-Seq. There are currently six Ns in it.

Adapter and primer sequences:

C1-P1-T31: 5'-Bio- AATGATACGGCGACCACCGATCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT -3'

P1B-UMI-RNA-TSO: 5'-Bio- CUACACGACGCUCUUCCGAUCU[6-bp UMI]rGrGrG -3'

DI-PCR-P1A: 5'-Bio- AATGATACGGCGACCACCGA -3'

DI-P1A-idx[1-32]-P1B: 5'-Bio- AATGATACGGCGACCACCGAGATCTACAC[5-bp well barcode]CTACACGACGCTCTTCCGATC -3'

Barcoded STRT-Tn5-Idx[1-96] top (including 19-bp Mosaic End (ME)): 5'- CAAGCAGAAGACGGCATACGA[8-bp subarray barcode]GCGTCAGATGTGTATAAGAGACAG -3'

Universal Tn5 bottom (STRT-TN5-U): 5'- CTGTCTCTTATACACATCTGACGC -3'

Annealed Tn5 oligos for Tn5 transposome assembly:

5'- CAAGCAGAAGACGGCATACGA[8-bp subarray barcode]GCGTCAGATGTGTATAAGAGACAG -3'
                                                      3'- TCTACACATATTCTCTGTC -5'

^*P1_2nd_PCR: 5'- AATGATACGGCGACCACCGAGATC -3'

^*P2_2nd_PCR: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

DI-Read1-Seq: 5'- ATGATACGGCGACCACCGAGATCTACACNNNNNNCTACACGACGCTCTTCCGATCT -3'

DI_idxP1A-Seq: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

* Recent sequencing libraries use Illumina P5/P7 adapters, instead of Solexa P1/P2 adapters.

Step-by-step library generation

(1) Anneal C1-P1-T31 to mRNA and reverse transcription using MMLV:


5'- XXXXXXXXXXXXXXXXXXXB(A)_n
                 <----NV(T)₃₁GCTAGCCACCAGCGGCATAGTAA -Bio-5'

(2) After reverse transcription, the terminal tranferase acitivity of MMLV add extra Cs:


5'- XXXXXXXXXXXXXXXXXXXB(A)_n
 CCCXXXXXXXXXXXXXXXXXXNV(T)₃₁GCTAGCCACCAGCGGCATAGTAA -Bio-5'

(3) Adding P1B-UMI-RNA-TSO for second strand synthesis:


5'-Bio- CUACACGACGCUCUUCCGAUCU[6-bp UMI]GGGXXXXXXXXXXXXXXXXXXXX(A)_n
                                        CCCXXXXXXXXXXXXXXXXXXXX(T)₃₁GCTAGCCACCAGCGGCATAGTAA -Bio-5'

(4) cDNA amplification using single DI-PCR-P1A and DI-P1A-idx[1-32]-P1B primers to index the wells within each subarray:


5'-Bio- AATGATACGGCGACCACCGAGATCTACAC[5-bp well barcode]CTACACGACGCTCTTCCGATC---->
                                               5'-Bio-  CUACACGACGCUCUUCCGAUCU[6-bp UMI]GGGXXXXX...XXXXXX(pA)CGATCGGTGGTCGCCGTATCATT
                                                        GATGTGCTGCGAGAAGGCTAGA[6-bp UMI]CCCXXXXX...XXXXXX(dT)GCTAGCCACCAGCGGCATAGTAA -Bio-5'
                                                                                                           <----AGCCACCAGCGGCATAGTAA -Bio-5'

(5) Amplified double stranded and indexed cDNA looks like this:


5'-Bio- AATGATACGGCGACCACCGAGATCTACAC[5-bp well barcode]CTACACGACGCTCTTCCGATCT[6-bp UMI]GGGXXX...XXX(pA)CGATCGGTGGTCGCCGTATCATT
        TTACTATGCCGCTGGTGGCTCTAGATGTG[5-bp well barcode]GATGTGCTGCGAGAAGGCTAGA[6-bp UMI]CCCXXX...XXX(dT)GCTAGCCACCAGCGGCATAGTAA -Bio-5'

(6) Homemade Tn5 homodimers using annealed Barcoded STRT-Tn5-Idx[1-96] top/bottom tagmentation on amplified cDNA (will create 9-bp gap):


Product 1 (5'-end of cDNA):

5'-Bio- AATGATACGGCGACCACCGAGATCTACAC[5-bp well barcode]CTACACGACGCTCTTCCGATCT[6-bp UMI]GGGXXX...XXX         CTGTCTCTTATACACATCT
        TTACTATGCCGCTGGTGGCTCTAGATGTG[5-bp well barcode]GATGTGCTGCGAGAAGGCTAGA[6-bp UMI]CCCXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCG[8-bp subarray barcode]AGCATACGGCAGAAGACGAAC -5'




Product 2 (3'-end of cDNA):

5'-Bio- AATGATACGGCGACCACCGATCG(dT)XXX...XXX         CTGTCTCTTATACACATCT
        TTACTATGCCGCTGGTGGCTAGC(pA)XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCG[8-bp subarray barcode]AGCATACGGCAGAAGACGAAC -5'

(7) Streptavidin binding and PvuI digestion (5'...CGATCG...3'):


Product 1 (5'-end of cDNA) will not be affected:

|--5'-Bio- AATGATACGGCGACCACCGAGATCTACAC[5-bp well barcode]CTACACGACGCTCTTCCGATCT[6-bp UMI]GGGXXX...XXXCTGTCTCTTATACACATCTGACGC[8-bp subarray barcode]TCGTATGCCGTCTTCTGCTTG
           TTACTATGCCGCTGGTGGCTCTAGATGTG[5-bp well barcode]GATGTGCTGCGAGAAGGCTAGA[6-bp UMI]CCCXXX...XXXGACAGAGAATATGTGTAGACTGCG[8-bp subarray barcode]AGCATACGGCAGAAGACGAAC -5'




Product 2 (3'-end of cDNA) will be cut by PvuI (this product is omitted later since it cannot be amplified):

|--5'-Bio- AATGATACGGCGACCACCGAT   CG(dT)XXX...XXX         CTGTCTCTTATACACATCT
           TTACTATGCCGCTGGTGGC   TAGC(pA)XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCG[8-bp subarray barcode]AGCATACGGCAGAAGACGAAC -5'

(8) Library amplification using P1_2nd_PCR and P2_2nd_PCR priers:


       5'- AATGATACGGCGACCACCGAGATC-------->
|--5'-Bio- AATGATACGGCGACCACCGAGATCTACAC[5-bp well barcode]CTACACGACGCTCTTCCGATCT[6-bp UMI]GGGXXX...XXXCTGTCTCTTATACACATCTGACGC[8-bp subarray barcode]TCGTATGCCGTCTTCTGCTTG
           TTACTATGCCGCTGGTGGCTCTAGATGTG[5-bp well barcode]GATGTGCTGCGAGAAGGCTAGA[6-bp UMI]CCCXXX...XXXGACAGAGAATATGTGTAGACTGCG[8-bp subarray barcode]AGCATACGGCAGAAGACGAAC -5'
                                                                                                                                         <---------TAGAGCATACGGCAGAAGACGAAC -5'

(9) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNCTACACGACGCTCTTCCGATCTNNNNNNGGGXXX...XXXCTGTCTCTTATACACATCTGACGCNNNNNNNNTCGTATGCCGTCTTCTGCTTG
    TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNGATGTGCTGCGAGAAGGCTAGANNNNNNCCCXXX...XXXGACAGAGAATATGTGTAGACTGCGNNNNNNNNAGCATACGGCAGAAGACGAAC -5'
        Solexa P1                 5bp                        UMI       cDNA           ME                 8bp         Solexa P2
                                  well                                                                 subarray
                                barcode                                                                barcode

Library sequencing:

(1) Add DI-Read1-Seq sequencing primer to sequence the first read (bottom strand as template, the first 6-bp of the read will be UMI):


5'-  ATGATACGGCGACCACCGAGATCTACACNNNNNCTACACGACGCTCTTCCGATCT-------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNGATGTGCTGCGAGAAGGCTAGANNNNNNCCCXXX...XXXGACAGAGAATATGTGTAGACTGCGNNNNNNNNAGCATACGGCAGAAGACGAAC -5'

(2) Add STRT-Tn5-U primer to sequence subarray barcodes (bottom strand as template):


                                                                          5'- CTGTCTCTTATACACATCTGACGC------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNGATGTGCTGCGAGAAGGCTAGANNNNNNCCCXXX...XXXGACAGAGAATATGTGTAGACTGCGNNNNNNNNAGCATACGGCAGAAGACGAAC -5'

(3) Add DI_idxP1A-Seq primer to sequence well barcodes (bottom strand as template):


5'- AATGATACGGCGACCACCGAGATCTACAC---->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNGATGTGCTGCGAGAAGGCTAGANNNNNNCCCXXX...XXXGACAGAGAATATGTGTAGACTGCGNNNNNNNNAGCATACGGCAGAAGACGAAC -5'

STRT-seq / STRT-seq-C1 / STRT-seq-2i

STRT-seq

Adapter and primer sequences:

Step-by-step library generation

(1) Anneal oligo-dTVN to mRNA and reverse transcription using MMLV:

(2) After reverse transcription, the terminal tranferase acitivity of MMLV add extra Cs:

(3) Adding Barcoded TSO for second strand synthesis:

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

(5) Streptavidin beads binding, and cDNA fragmentation using DNase I in the presence of Mn2+, which preferentially causes double-stranded breaks:

(6) A tailing:

(7) Ligation with doube stranded P2 adapter with T overhang, and at the same time, SalI digestion (5'...GTCGAC...3'):

(8) Add Library PCR Primers 1&2 for amplification:

(9) Final library structure:

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, the first 6-bp of the read will be the cell barcode):

(2) Cluster regeneration, add read 2 sequencing primer to sequence the second read (top strand as template, not sure whether this step is actually perfromed):

STRT-seq-C1

Adapter and primer sequences:

Step-by-step library generation

(1) Anneal C1-P1-T31 to mRNA and reverse transcription using MMLV:

(2) After reverse transcription, the terminal tranferase acitivity of MMLV add extra Cs:

(3) Adding C1-P1-RNA-TSO for second strand synthesis:

(4) cDNA amplification using single C1-P1-PCR-2 primer:( i.e. semi-suppressive PCR )

(5) Homemade Tn5 homodimers using C1-Tn5_top/bottom tagmentation on amplified cDNA (will create 9-bp gap):

(6) Streptavidin beads binding. and PvuI cleavage (5'...CGATCG...3')

(7) NaOH denature Product 1 (5'-end of cDNA), and library amplification with P1 and P2 adapters using bottom strand as template:

(8) Final library structure:

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, the first 5-bp of the read will be UMI):

(2) Add index sequencing primer to sequence cell barcodes (bottom strand as template):

STRT-seq-2i

Adapter and primer sequences:

Step-by-step library generation

(1) Anneal C1-P1-T31 to mRNA and reverse transcription using MMLV:

(2) After reverse transcription, the terminal tranferase acitivity of MMLV add extra Cs:

(3) Adding P1B-UMI-RNA-TSO for second strand synthesis:

(4) cDNA amplification using single DI-PCR-P1A and DI-P1A-idx[1-32]-P1B primers to index the wells within each subarray:

(5) Amplified double stranded and indexed cDNA looks like this:

(6) Homemade Tn5 homodimers using annealed Barcoded STRT-Tn5-Idx[1-96] top/bottom tagmentation on amplified cDNA (will create 9-bp gap):

(7) Streptavidin binding and PvuI digestion (5'...CGATCG...3'):

(8) Library amplification using P1_2nd_PCR and P2_2nd_PCR priers:

(9) Final library structure:

Library sequencing:

(1) Add DI-Read1-Seq sequencing primer to sequence the first read (bottom strand as template, the first 6-bp of the read will be UMI):

(2) Add STRT-Tn5-U primer to sequence subarray barcodes (bottom strand as template):

(3) Add DI_idxP1A-Seq primer to sequence well barcodes (bottom strand as template):

(5) Streptavidin beads binding, and cDNA fragmentation using DNase I in the presence of Mn²⁺, which preferentially causes double-stranded breaks: