The sci-RNA-seq uses the combinatorial indexing to identify single cells without single cell isolation. Two-level indexing (RT barcode + PCR barcodes (i5 + i7)) or three-level indexing (RT barcode + PCR barcodes (i5 + i7) + Tn5 barcodes) can be used. Three-level indexing is a bit more difficult since you need to assemble many indexed Tn5 transposomes. Here, two-level indexing strategy is demonstrated.
Barcoded RT primer: 5'-
Nextera Tn5 binding site (19-bp Mosaic End (ME)): 5'-
Nextera N/S5xx primer entry point (s5): 5'-
Nextera N7xx primer entry point (s7): 5'-
Illumina P5 Primer: 5'-
Illumina P7 Primer: 5'-
Read 1 sequencing primer: 5'-
Index 1 sequencing primer (i7): 5'-
Index 2 sequencing primer (i5): 5'-
Read 2 seuquencing primer: 5'-
5'- ACGACGCTCTTCCGATCT [8-bp UMI] [10-bp RT barcode] (T)30VN----------> (A)n BXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX -5'
5'- ACGACGCTCTTCCGATCT [8-bp UMI] [10-bp RT barcode] (dT)VXXXXXXXXXXXXXXXXXXXXXXX -3' 3'-TGCTGCGAGAAGGCTAGA [8-bp UMI] [10-bp RT barcode] (pA)BXXXXXXXXXXXXXXXXXXXXXXX -5'
Product 1 (s5 at both ends, not amplifiable due to the use of Illumina P5/P7 Primer, see the next step): 5'- TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT -5' Product 2 (s7 at both ends, not amplifiable due to the use of Illumina P5/P7 Primer, see the next step): 5'-GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' Product 3 (different s5 and s7 at both ends, not amplifiable, due to the use of Illumina P5/P7 Primer, see the next step): 5'-TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' Product 4 (s5 at one end, 3' of cDNA at the other end, not amplifiable, due to the use of Illumina P5/P7 Primer, see the next step): 5'-ACGACGCTCTTCCGATCT [8-bp UMI] [10-bp RT barcode] (dT)VXXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT -3' 3'-TGCTGCGAGAAGGCTAGA [8-bp UMI] [10-bp RT barcode] (pA)BXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT -5' Product 5 (s7 at one end, 3' of cDNA at the other end, the only amplifiable product, see the next step): 5'-ACGACGCTCTTCCGATCT [8-bp UMI] [10-bp RT barcode] (dT)VXXX...XXXCTGTCTCTTATACACATCT -3' 3'-TGCTGCGAGAAGGCTAGA [8-bp UMI] [10-bp RT barcode] (pA)BXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
5'- ACGACGCTCTTCCGATCT [8-bp UMI] [10-bp RT barcode] (dT)VXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC -3' 3'-TGCTGCGAGAAGGCTAGA [8-bp UMI] [10-bp RT barcode] (pA)BXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
5'- AATGATACGGCGACCACCGAGATCTACAC [i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCT ------> 5'-ACGACGCTCTTCCGATCT [8-bp UMI] [10-bp RT barcode] (dT)VXXX...XXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC -3' 3'-TGCTGCGAGAAGGCTAGA [8-bp UMI] [10-bp RT barcode] (pA)BXXX...XXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' <---------GGCTCGGGTGCTCTG [i7]TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNNN NNNNNNNNNN (dT)VXXX...XXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNNNN NNNNNNNNNN (pA)BXXX...XXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 i5This bit is Truseq adapter 8bp UMI 10bp RT cDNAME s7 i7Illumina P7 barcode
5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT -----------------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNNNN NNNNNNNNNN (pA)BXXX...XXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC ---------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNNNN NNNNNNNNNN (pA)BXXX...XXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNNN NNNNNNNNNN (dT)VXXX...XXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <--------TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -5'
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNNN NNNNNNNNNN (dT)VXXX...XXXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <-----GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'