FLASH-seq

The protocols of SMART-seq and SMART-seq2 are almost the same. SMART-seq2 is an improved version of SMART-seq. The authors performed 457 optimisation experiments to test conditions. Two key parameters are:

(1) exchanging the last guanylate for a locked nucleic acid (LNA) at the 3' end of TSO.

(2) Include methyl group donor betaine in combination with higher MgCl₂ concentrations.

SMART-seq3 is a further improvement comparing to SMART-seq/SMART-seq2, and it has a different oligo design compared to SMART-seq/SMART-seq2. Major improvements include:

(1) Use Maxima H- reverse transcriptase.

(2) Use NaCl (instead of the common KCl) during reverse transcription.

(3) Use 5% PEG as a crowding reagent during reverse transcription, like mcSCRB-seq.

(4) Use a unique 11-bp tag and UMI in the TSO so that the 5' read can be distinguished from the internal reads.

Built upon SMART-seq3, SMART-seq3xpress and FLASH-seq made some further improvements in terms of the processing speed and reagent cost by removing some intermediate purification steps and reduce the reaction volume to nanolitre scale.

SMART-seq / SMART-seq2

Adapter and primer sequences:

oligo-dTVN: 5'- AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN -3'

Template Switching Oligo (TSO): 5'- AAGCAGTGGTATCAACGCAGAGTACATrGrG+G -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'

Nextera (XT) N/S5xx Index primer: 5'- AATGATACGGCGACCACCGAGATCTACAC[8-bp i5 index]TCGTCGGCAGCGTC -3'

Nextera (XT) N7xx Index primer: 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp i7 index]GTCTCGTGGGCTCGG -3'

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

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

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

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

8-bp i5 & i7 sequences:

    N/S502 : CTCTCTAT
    N/S503 : TATCCTCT
    N/S505 : GTAAGGAG
    N/S506 : ACTGCATA
    N/S507 : AAGGAGTA
    N/S508 : CTAAGCCT
    N/S510 : CGTCTAAT
    N/S511 : TCTCTCCG
    N/S513 : TCGACTAG
    N/S515 : TTCTAGCT
    N/S516 : CCTAGAGT
    N/S517 : GCGTAAGA
    N/S518 : CTATTAAG
    N/S520 : AAGGCTAT
    N/S521 : GAGCCTTA
    N/S522 : TTATGCGA

    N701 : TCGCCTTA
    N702 : CTAGTACG
    N703 : TTCTGCCT
    N704 : GCTCAGGA
    N705 : AGGAGTCC
    N706 : CATGCCTA
    N707 : GTAGAGAG
    N710 : CAGCCTCG
    N711 : TGCCTCTT
    N712 : TCCTCTAC
    N714 : TCATGAGC
    N715 : CCTGAGAT
    N716 : TAGCGAGT
    N718 : GTAGCTCC
    N719 : TACTACGC
    N720 : AGGCTCCG
    N721 : GCAGCGTA
    N722 : CTGCGCAT
    N723 : GAGCGCTA
    N724 : CGCTCAGT
    N726 : GTCTTAGG
    N727 : ACTGATCG
    N728 : TAGCTGCA
    N729 : GACGTCGA

Step-by-step library generation

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


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

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


5'- XXXXXXXXXXXXXXXXXXXXB(A)_n
 CCCXXXXXXXXXXXXXXXXXXXNV(T)₃₀CATGAGACGCAACTATGGTGACGAA -5'

(3) Adding TSO for second strand synthesis:


5'- AAGCAGTGGTATCAACGCAGAGTACATGGGXXXXXXXXXXXXXXXXXXXX(A)_n
                        <------CCCXXXXXXXXXXXXXXXXXXXX(T)₃₀CATGAGACGCAACTATGGTGACGAA -5'

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


5'- AAGCAGTGGTATCAACGCAGAGT------>
5'- AAGCAGTGGTATCAACGCAGAGTACATGGGXXXXXXX...XXXXXXX(pA)GTACTCTGCGTTGATACCACTGCTT
    TTCGTCACCATAGTTGCGTCTCATGTACCCXXXXXXX...XXXXXXX(dT)CATGAGACGCAACTATGGTGACGAA -5'
                                                  <------TGAGACGCAACTATGGTGACGAA -5'

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


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

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


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

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


Product 3 (different ends, amplifiable):

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

(6) 72 degree gap fill-in (the first cycle in Nextera PCR):


5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC
    AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(7) Amplification using N/S5xx and N7xx index primers:


5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC---->
                                 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC
                                     AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                               <----GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'

(8) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
    TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
           Illumina P5              i5         s5              ME                cDNA                ME               s7          i7            Illumina P7

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template):


                                     5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG------>
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Index 1 sequencing primer to sequence the first index (i7) (bottom strand as template):


                                                                                         5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC------>
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add Index 2 sequencing primer to sequence the seconde index (i5) (top strand as template):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
                                 <-------AGCAGCCGTCGCAGTCTACACATATTCTCTGTC -5'

(4) Add read 2 sequencing primer to sequence the second read (top strand as template):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
                                                                                      <------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

SMART-seq3 / SMART-seq3xpress / FLASH-seq

NOTE: These three methods differ on the experimental details of how the protocol is executed. The library generation procedures are the same, and the final libraries are ALMOST the same. The only differences are that they used different variation of TSO, which makes the 5' fragment a bit different. Here, only SMART-seq3 procedures are shown. The final libraries of these three methods are shown at the end of the workflow.

Adapter and primer sequences:

Smartseq3_OligodT30VN: 5'- /5Biosg/ACGAGCATCAGCAGCATACGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN -3'

Smartseq3_N8_TSO: 5'- /5Biosg/AGAGACAGATTGCGCAATG[8bp UMI]rGrGrG -3'

Smartseq3xpress_TSO: 5'- /5Biosg/AGAGACAGATTGCGCAATG[8bp UMI]WWrGrGrG -3'

FLASH-seq_TSO: 5'- /5Biosg/AGAGACAGATTGCGCAATG[8bp UMI]CTAACrGrGrG -3'

Fwd_PCR_primer: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAA*T*G -3'

Rev_PCR_primer: 5'- ACGAGCATCAGCAGCATAC*G*A -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'

Nextera (XT) N/S5xx Index primer: 5'- AATGATACGGCGACCACCGAGATCTACAC[8-bp i5 index]TCGTCGGCAGCGTC -3'

Nextera (XT) N7xx Index primer: 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp i7 index]GTCTCGTGGGCTCGG -3'

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

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

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

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

Step-by-step library generation

(1) Anneal Smartseq3_OligodT30VN to mRNA and reverse transcription using Maxima H- MMLV:


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

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


5'- XXXXXXXXXXXXXXXXXXXXB(A)_n
 CCCXXXXXXXXXXXXXXXXXXXNV(T)₃₀AGCATACGACGACTACGAGCA -5'

(3) TSO anneals to the extra Cs for template switching:


5'- AGAGACAGATTGCGCAATG[8bp UMI]GGGXXXXXXXXXXXXXXXXXXXX(A)_n
                         <------CCCXXXXXXXXXXXXXXXXXXXX(T)₃₀AGCATACGACGACTACGAGCA -5'

(4) Adding Fwd_PCR_primer and Rev_PCR_primer for cDNA amplification:


5′- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG------>
                         5'- AGAGACAGATTGCGCAATG[8bp UMI]GGGXXXXXXXXXXXXXXXXXXXX(pA)TCGTATGCTGCTGATGCTCGT
                             TCTCTGTCTAACGCGTTAC[8bp UMI]CCCXXXXXXXXXXXXXXXXXXXX(dT)AGCATACGACGACTACGAGCA -5'
                                                                             <------AGCATACGACGACTACGAGCA -5'

(5) Purify amplified cDNA:


5′- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG[8bp UMI]GGGXXXXXXXXXXXXXXXXXXXX(pA)TCGTATGCTGCTGATGCTCGT -3'
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTCTAACGCGTTAC[8bp UMI]CCCXXXXXXXXXXXXXXXXXXXX(dT)AGCATACGACGACTACGAGCA -5'

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


Product 1 (5' fragments, s5 at both ends, not amplifiable due to semi-suppressive PCR):

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG[8bp UMI]GGGXXX...XXX         CTGTCTCTTATACACATCT
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTCTAACGCGTTAC[8bp UMI]CCCXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'


Product 2 (5' fragments, different ends, this is amplifiable):

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG[8bp UMI]GGGXXX...XXX         CTGTCTCTTATACACATCT
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTCTAACGCGTTAC[8bp UMI]CCCXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'


Product 3 (internal fragments, s5 at both ends, not amplifiable due to semi-suppressive PCR):

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


Product 4 (internal fragments, s7 at both ends, not amplifiable due to semi-suppressive PCR):

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


Product 5 (internal fragments, different ends, this is amplifiable):

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

(7) 72 degree gap fill-in (the first cycle in Nextera PCR):


5' fragments:

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG[8bp UMI]GGGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTCTAACGCGTTAC[8bp UMI]CCCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

Internal fragments:

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC
    AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(8) Amplification using N/S5xx and N7xx index primers:


5' fragments:


5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC---->
                                 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG[8bp UMI]GGGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                                 3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTCTAACGCGTTAC[8bp UMI]CCCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                                                      <----GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'


Internal fragments:


5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC---->
                                 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC
                                     AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                               <----GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'

(9) Final library structure:

(9.1) 5' Fragments


SMART-seq3:

5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATGNNNNNNNNGGGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCTAACGCGTTACNNNNNNNNCCCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
           Illumina P5              i5         s5              ME            11 bp     8 bp       cDNA           ME               s7          i7            Illumina P7
                                                                         5' fragment   UMI
                                                                              tag


SMART-seq3xpress:

5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATGNNNNNNNNWWGGGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCTAACGCGTTACNNNNNNNNSSCCCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
           Illumina P5              i5         s5              ME            11 bp     8 bp         cDNA           ME               s7          i7            Illumina P7
                                                                         5' fragment   UMI
                                                                              tag


FLASH-seq:

5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATGNNNNNNNNCTAACGGGXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCTAACGCGTTACNNNNNNNNGATTGCCCXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
           Illumina P5              i5         s5              ME            11 bp     8 bp            cDNA           ME               s7          i7            Illumina P7
                                                                         5' fragment   UMI
                                                                              tag

(9.2) Internal fragments


All three methods are the same:

5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
    TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
           Illumina P5              i5         s5              ME                cDNA                ME               s7          i7            Illumina P7

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, if read1 contains the 11bp 5' fragment tags, then the read pair is from the 5' of the transcript):


                                     5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG------>
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Index 1 sequencing primer to sequence the first index (i7) (bottom strand as template):


                                                                                         5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC------>
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add Index 2 sequencing primer to sequence the second index (i5) (top strand as template, single cells can be identified by the combination of i5 and i7):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
                                 <-------AGCAGCCGTCGCAGTCTACACATATTCTCTGTC -5'

(4) Add read 2 sequencing primer to sequence the second read (top strand as template):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
                                                                                      <------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'