SPLiT-seq / microSPLiT

The SPLiT-seq uses the combinatorial indexing to identify single cells without single cell isolation. Multi-level indexing can be performed by ligations. The workflow described here is based on the publication of Science 360, 176-182 (2018). To be consistent with the publication, all the oligo/primer names are the same as the Science 2018 publication. All oligos can be found in the Supplementary Table S12 from the publication. NOTE: The published version in this page is different from the preprint version in the bioRxiv. I have archived the previous Github Page about SPLiT-seq, which was based on the preprint version. If you want to have a look, you can check by clicking here.

Be careful that there are different versions of the protocol, and they use slightly different oligo sequences. The basic idea of the method does not change. Check this thread for more information.

microSPLiT was based on SPLiT-seq, and it was developed from the same lab that developed SPLiT-seq. microSPLiT was further optimised to work on sequencing mRNA from bacteria. The oligo sequences used in microSPLiT is almost the same as those in SPLiT-seq, except that the Round2 linker region is shorter. Therefore, the final library of microSPLiT is extremely similar to the original SPLiT-seq. The procedures described here is based on the Science paper and the oligo sequences are taken from their Supplementary Table S3.

SPLiT-seq

Adapter and primer sequences:

Oligo-dTVN (Round1_01-48): 5'-/5Phos/ AGGCCAGAGCATTCG[8-bp Round1 barcode]TTTTTTTTTTTTTTTVN -3'

Oligo-randN (Round1_49-96): 5'-/5Phos/ AGGCCAGAGCATTCG[8-bp Round1 barcode]NNNNNN -3'

Round2 Ligation Barcodes (Round2_01-96): 5'-/5Phos/ CATCGGCGTACGACT[8-bp Round2 barcode]ATCCACGTGCTTGAG -3'

Round2 Barcode Linker (BC_0215): 5'- CGAATGCTCTGGCCTCTCAAGCACGTGGAT -3'

Round3 Ligation Barcodes (Round3_01-96): 5'-/5Biosg/ CAGACGTGTGCTCTTCCGATCT[10-bp UMI][8-bp Round3 barcode]GTGGCCGATGTTTCG -3'

Round3 Barcode Linker (BC_0060): 5'- AGTCGTACGCCGATGCGAAACATCGGCCAC -3'

Round2 blocking strand (BC_0216): 5'- ATCCACGTGCTTGAGCGCGCTGCATACTTG -3'

Round3 blocking strand (BC_0066): 5'- GTGGCCGATGTTTCGCATCGGCGTACGACT -3'

Template Switching Oligos (TSO, BC_0127): 5'- AAGCAGTGGTATCAACGCAGAGTGAATrGrG+G -3'

cDNA Amplification Primer 1 (BC_0062): 5'- CAGACGTGTGCTCTTCCGATCT -3'

cDNA Amplification Primer 2 (BC_0108): 5'- AAGCAGTGGTATCAACGCAGAGT -3'

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

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

Indexed Library PCR Primer 1 (BC_0076 - BC_0083) : 5'- CAAGCAGAAGACGGCATACGAGAT[6-bp i7 sample index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3'

Library PCR Primer 2 (BC_0118): 5'- AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'

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

Index 1 sequencing primer: 5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'

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

Step-by-step library generation

(1) Prepare ligation adapters by annealing barcodes with corresponding linkers:


Round2 adapters: anneal Round2 Ligation Barcodes (Round2_01-96) with Round2 Barcode Linker (BC_0215):

5'- CGAATGCTCTGGCCTCTCAAGCACGTGGAT
                   GAGTTCGTGCACCTA[8-bp Round2 barcode]TCAGCATGCGGCTAC /5Phos/-5'


Round3 adapters: anneal Round3 Ligation Barcodes (Round3_01-96) with Round3 Barcode Linker (BC_0060):

5'- AGTCGTACGCCGATGCGAAACATCGGCCAC
                   GCTTTGTAGCCGGTG[8-bp Round3 barcode][10-bp UMI]TCTAGCCTTCTCGTGTGCAGAC /5Biosg/-5'

(2) Anneal Oligo-dTVN and Oligo-randN primers to mRNA and reverse transcription using Maxima H Minus Reverse Transcriptase in situ to add Round 1 barcodes:


mRNA 3':

5'- XXX...XXXB(A)_n
        <---NV(T)₁₅[8-bp Round 1 barcode]GCTTACGAGACCGGA /5Phos/-5'

mRNA internal (omitted in the rest stages):

5'- XXX...XXX(A)_n
    <-NNNNNN[8-bp Round 1 barcode]GCTTACGAGACCGGA /5Phos/-5'

(3) Pool and split to the plate containing Round2 adapters to add Round 2 barcodes:


5'- XXX...XXXB(pA)                     CGAATGCTCTGGCCTCTCAAGCACGTGGAT
 CCCXXX...XXXV(dT)[8-bp Round1 barcode]GCTTACGAGACCGGAGAGTTCGTGCACCTA[8-bp Round2 barcode]TCAGCATGCGGCTAC /5Phos/-5'

(4) Pool and split to the plate containing Round3 adapters to add Round 3 barcodes:


5'- XXX...XXXB(pA)                     CGAATGCTCTGGCCTCTCAAGCACGTGGAT                     AGTCGTACGCCGATGCGAAACATCGGCCAC
 CCCXXX...XXXV(dT)[8-bp Round1 barcode]GCTTACGAGACCGGAGAGTTCGTGCACCTA[8-bp Round2 barcode]TCAGCATGCGGCTACGCTTTGTAGCCGGTG[8-bp Round3 barcode][10-bp UMI]TCTAGCCTTCTCGTGTGCAGAC /5Biosg/-5'

(5) Pool, count cells, split into sublibraries. Then, for each sublibrary, perform cell lysis, low temperature (55 degree celcius) reverse crosslink and bind cDNA to streptavidin beads


3'- CCCXXX...XXXV(dT)[8-bp Round1 barcode]GCTTACGAGACCGGAGAGTTCGTGCACCTA[8-bp Round2 barcode]TCAGCATGCGGCTACGCTTTGTAGCCGGTG[8-bp Round3 barcode][10-bp UMI]TCTAGCCTTCTCGTGTGCAGAC /5Biosg/-5'

(6) Add TSO (BC_0127) to each sublibrary for second strand synthesis:


5'- AAGCAGTGGTATCAACGCAGAGTGAATGGG---->
                           <---CCCXXX...XXXV(dT)[8-bp Round1 barcode]GCTTACGAGACCGGAGAGTTCGTGCACCTA[8-bp Round2 barcode]TCAGCATGCGGCTACGCTTTGTAGCCGGTG[8-bp Round3 barcode][10-bp UMI]TCTAGCCTTCTCGTGTGCAGAC /5Biosg/-5'--|

(7) Add cDNA Amplification Primers 1 & 2 (BC_0062 and BC_0108) to each sublibrary:


5'- AAGCAGTGGTATCAACGCAGAGT------>
5'- AAGCAGTGGTATCAACGCAGAGTGAATGGGXXX...XXXB(pA)[8-bp Round1 barcode]CGAATGCTCTGGCCTCTCAAGCACGTGGAT[8-bp Round2 barcode]AGTCGTACGCCGATGCGAAACATCGGCCAC[8-bp Round3 barcode][10-bp UMI]AGATCGGAAGAGCACACGTCTG
    TTCGTCACCATAGTTGCGTCTCACTTACCCXXX...XXXV(dT)[8-bp Round1 barcode]GCTTACGAGACCGGAGAGTTCGTGCACCTA[8-bp Round2 barcode]TCAGCATGCGGCTACGCTTTGTAGCCGGTG[8-bp Round3 barcode][10-bp UMI]TCTAGCCTTCTCGTGTGCAGAC /5Biosg/-5'--|
                                                                                                                                                                               <------TCTAGCCTTCTCGTGTGCAGAC -5'

(8) Tagmentation with Illumina Nextera XT Kit:


Product 1 (s5 at both ends, not amplifiable due to PCR primers used, see the next step):

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



Product 2 (s7 at both ends, not amplifiable due to PCR primers used, see the next step):

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



Product 3 (different s5 and s7 at both ends, not amplifiable, due to PCR primers used, see the next step):

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


Product 4 (s7 at one end, 3' of cDNA at the other end, not amplifiable due to PCR primers used, see the next step):

5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX(pA)[8-bp Round1 barcode]CGAATGCTCTGGCCTCTCAAGCACGTGGAT[8-bp Round2 barcode]AGTCGTACGCCGATGCGAAACATCGGCCAC[8-bp Round3 barcode][10-bp UMI]AGATCGGAAGAGCACACGTCTG -3'
                   TCTACACATATTCTCTGTC         XXX...XXX(dT)[8-bp Round1 barcode]GCTTACGAGACCGGAGAGTTCGTGCACCTA[8-bp Round2 barcode]TCAGCATGCGGCTACGCTTTGTAGCCGGTG[8-bp Round3 barcode][10-bp UMI]TCTAGCCTTCTCGTGTGCAGAC -5'


Product 5 (s5 at one end, 3' of cDNA at the other end, the only amplifiable fragment, see the next step):

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX(pA)[8-bp Round1 barcode]CGAATGCTCTGGCCTCTCAAGCACGTGGAT[8-bp Round2 barcode]AGTCGTACGCCGATGCGAAACATCGGCCAC[8-bp Round3 barcode][10-bp UMI]AGATCGGAAGAGCACACGTCTG -3'
                  TCTACACATATTCTCTGTC         XXX...XXX(dT)[8-bp Round1 barcode]GCTTACGAGACCGGAGAGTTCGTGCACCTA[8-bp Round2 barcode]TCAGCATGCGGCTACGCTTTGTAGCCGGTG[8-bp Round3 barcode][10-bp UMI]TCTAGCCTTCTCGTGTGCAGAC -5'

(9) Add Library PCR Primer 1 (one of BC_0076 - BC_0083) and 2 (BC_0018) to index each sublibrary:


5'- AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG------->
                                     5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX(pA)[8-bp Round1 barcode]CGAATGCTCTGGCCTCTCAAGCACGTGGAT[8-bp Round2 barcode]AGTCGTACGCCGATGCGAAACATCGGCCAC[8-bp Round3 barcode][10-bp UMI]AGATCGGAAGAGCACACGTCTG -3'
                                                       TCTACACATATTCTCTGTC         XXX...XXX(dT)[8-bp Round1 barcode]GCTTACGAGACCGGAGAGTTCGTGCACCTA[8-bp Round2 barcode]TCAGCATGCGGCTACGCTTTGTAGCCGGTG[8-bp Round3 barcode][10-bp UMI]TCTAGCCTTCTCGTGTGCAGAC -5'
                                                                                                                                                                                                                             <--------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'

(10) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXX(pA)NNNNNNNNCGAATGCTCTGGCCTCTCAAGCACGTGGATNNNNNNNNAGTCGTACGCCGATGCGAAACATCGGCCACNNNNNNNNNNNNNNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGATCTAGCGAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXX(dT)NNNNNNNNGCTTACGAGACCGGAGAGTTCGTGCACCTANNNNNNNNTCAGCATGCGGCTACGCTTTGTAGCCGGTGNNNNNNNNNNNNNNNNNNTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
             Illumina P5                       s5               ME          cDNA         8 bp           Round2 linker          8 bp          Round3 linker           8 bp    10 bp             TruSeq Read 2           6bp i7      Illumina P7
                                                                                        Round1                                Round2                                Round3    UMI
                                                                                        Barcode                               Barcode                               Barcode

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, cDNA reads, 66 cycles):


                                     5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG----------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGATCTAGCGAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXX(dT)NNNNNNNNGCTTACGAGACCGGAGAGTTCGTGCACCTANNNNNNNNTCAGCATGCGGCTACGCTTTGTAGCCGGTGNNNNNNNNNNNNNNNNNNTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Index 1 sequencing primer to sequence i7 index (bottom strand as template, 6 cycles):


                                                                                                                                                                                  5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC----->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGATCTAGCGAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXX(dT)NNNNNNNNGCTTACGAGACCGGAGAGTTCGTGCACCTANNNNNNNNTCAGCATGCGGCTACGCTTTGTAGCCGGTGNNNNNNNNNNNNNNNNNNTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add Read 2 sequencing primer to sequence the second read (top strand as template, UMI and 3 rounds of barcodes, 94 cycles):


5'- AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXX(pA)NNNNNNNNCGAATGCTCTGGCCTCTCAAGCACGTGGATNNNNNNNNAGTCGTACGCCGATGCGAAACATCGGCCACNNNNNNNNNNNNNNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                       <---------------------------------------------------------------------------------------------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'

microSPLiT

Adapter and primer sequences:

Oligo-dTVN (Round1_01-48): 5'-/5Phos/ ACTGTGG[8-bp Round1 barcode]TTTTTTTTTTTTTTTVN -3'

Oligo-randN (Round1_49-96): 5'-/5Phos/ ACTGTGG[8-bp Round1 barcode]NNNNNN -3'

Round2 Ligation Barcodes (Round2_01-96): 5'-/5Phos/ CATCGGCGTACGACT[8-bp Round2 barcode]ATCCACGTGCTTGAG -3'

Round2 Barcode Linker (BC_0335): 5'- CCACAGTCTCAAGCACG -3'

Round3 Ligation Barcodes (Round3_01-96): 5'-/5Biosg/ CAGACGTGTGCTCTTCCGATCT[10-bp UMI][8-bp Round3 barcode]GTGGCCGATGTTTCG -3'

Round3 Barcode Linker (BC_0284): 5'- TACGCCGATGCGAAACATCG -3'

Round2 blocking strand (BC_0340): 5'- CGTGCTTGAGACTGTGG -3'