SCRB-seq / mcSCRB-seq

mcSCRB-seq is an improvement over the original SCRB-seq by using a crowding reagent during RT and other stuff. Check the manuscripts for more details. The library construction strategy is the same.

Adapter and primer sequences:

E3V6NEXT primer: 5'-/5Biosg/ACACTCTTTCCCTACACGACGCTCTTCCGATCT[6-bp cell barcode][10-bp UMI]T₃₀VN -3'

E5V6NEXT primer: 5'- iCiGiCACACTCTTTCCCTACACGACGCrGrGrG -3'

SINGV6 primer: 5'- /5Biosg/ACACTCTTTCCCTACACGACGC -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'

P5NEXTPT5 primer: 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG*A*T*C*T* -3'

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

TruSeq Read 1 sequencing primer: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT -3'

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

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

Step-by-step library generation

(1) Anneal E3V6NEXT primer to mRNA and reverse transcription using MMLV:


5'- XXXXXXXXXXXXXXXXXXXB(A)_n
                 <----NV(T)₃₀[10-bp UMI][6-bp cell barcode]TCTAGCCTTCTCGCAGCACATCCCTTTCTCACA -5'

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


5'- XXXXXXXXXXXXXXXXXXXXB(A)_n
 CCCXXXXXXXXXXXXXXXXXXXNV(T)₃₀[10-bp UMI][6-bp cell barcode]TCTAGCCTTCTCGCAGCACATCCCTTTCTCACA -5'

(3) Adding E5V6NEXT primer for template switching and second strand synthesis:


5'- CGCACACTCTTTCCCTACACGACGCGGGXXXXXXXXXXXXXXXXXXXX(A)_n------>
                      <------CCCXXXXXXXXXXXXXXXXXXXX(T)₃₀[10-bp UMI][6-bp cell barcode]TCTAGCCTTCTCGCAGCACATCCCTTTCTCACA -5'

(4) Adding SINGV6 primer for single primer cDNA amplification:( i.e. semi-suppressive PCR )


   5'- ACACTCTTTCCCTACACGACGC------>
5'- CGCACACTCTTTCCCTACACGACGCGGGXXXXXXXXXXXXXXXXXXXX(pA)[10-bp UMI][6-bp cell barcode]AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
    GCGTGTGAGAAAGGGATGTGCTGCGCCCXXXXXXXXXXXXXXXXXXXX(dT)[10-bp UMI][6-bp cell barcode]TCTAGCCTTCTCGCAGCACATCCCTTTCTCACA -5'
                                                                                          <------CGCAGCACATCCCTTTCTCACA -5'

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


Product 1, 2 & 3 (middle of cDNA, Nextera adapter sequence (s5 or s7) at the ends, not amplifiable due to primer used, see the next step):

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


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


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


Product 4 & 5 (5' of cDNA, s5 or s7 at the other end, not amplifiable due to primer used, see the next step):

5'- CGCACACTCTTTCCCTACACGACGCGGGXXXXXXXXXXX...XXXXXXXXX         CTGTCTCTTATACACATCT
    GCGTGTGAGAAAGGGATGTGCTGCGCCCXXXXXXXXXXX...XXXXXXXXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

5'- CGCACACTCTTTCCCTACACGACGCGGGXXXXXXXXXXX...XXXXXXXXX         CTGTCTCTTATACACATCT
    GCGTGTGAGAAAGGGATGTGCTGCGCCCXXXXXXXXXXX...XXXXXXXXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'


Product 6 (3' of cDNA, s5 at the other end, not amplifiable due to primer used, see the next step):

5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT[6-bp cell barcode][10-bp UMI](dT)XXXXXX...XXXXXX         CTGTCTCTTATACACATCT
    TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[6-bp cell barcode][10-bp UMI](pA)XXXXXX...XXXXXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'


Product 7 (3' of cDNA, s7 at the other end, the only amplifiable fragment, see the next step):

5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT[6-bp cell barcode][10-bp UMI](dT)XXXXXX...XXXXXX         CTGTCTCTTATACACATCT
    TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[6-bp cell barcode][10-bp UMI](pA)XXXXXX...XXXXXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(6) Amplification using P5NEXTPT5 primer and Nextera N7xx index primers:


5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT---->
                         5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT[6-bp cell barcode][10-bp UMI](dT)XXXXXX...XXXXXX         CTGTCTCTTATACACATCT
                             TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[6-bp cell barcode][10-bp UMI](pA)XXXXXX...XXXXXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                                                      <----GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'

(7) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNN(dT)XXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNN(pA)XXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
           Illumina P5                  TruSeq Read 1          6bp   10bp UMI       cDNA           ME               s7          i7          Illumina P7
                                                               cell
                                                             barcode

Library sequencing:

(1) Add TruSeq Read 1 primer to sequence the first read (bottom strand as template, this is the cell barcode and UMI):


                         5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT--------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNN(pA)XXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

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


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

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


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