The Chromium Single Cell 5’ Gene Expression is shown here. Oligo sequence information is taken from The 10x Genomics Technical Note. The 5' Gene Expression kit is similar to the 3' kit. Instead of using barcoded RT primers on the beads, the 5' Gene Expression kit use the same RT primer, but with barcoded Template Swithcing Oligos (TSO) on their gel beads. The 5' Gene Expression kit is often coupled with profiling V(D)J from T/B cells (with TCR/BCR primers), but you can simply use this kit for gene expression purposes. Conceptually, this kit is very similar to STRT-seq.
Beads-TSO (PN-220112): |--5'-
Poly-dT RT primer (PN-2000007): 5'-
cDNA Primer Mix (for cDNA amplification, PN-220106):
Forward primer: 5'- CTACACGACGCTCTTCCGATCT -3' Reverse primer: 5'-AAGCAGTGGTATCAACGCAG -3'
Illumina Truseq Read 1 primer: 5'-
Illumina Truseq Read 2 primer: 5'-
Truseq adapter (double stranded DNA with a T overhang, PN-220026):
5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3' 3'-TCTAGCCTTCTCG -5'
Library PCR primer 1 (PN-220111): 5'-
Library PCR primer 2 (PN-220103): 5'-
Sample index sequencing primer: 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
<--------NV(T)30 CATGAGACGCAACTATGGTGACGAA -5' 5'- XXXXXXXXXXXXXXXXXXXXXB(A)30
CCCXXXXXXXXXXXXXXXXXXXXXNV(T)30 CATGAGACGCAACTATGGTGACGAA -5' 5'- XXXXXXXXXXXXXXXXXXXXXXB(A)30
<-----------CCCXXXXXXXXXXXXXXXXXXXXXNV(T)30 CATGAGACGCAACTATGGTGACGAA -5' |--5'-CTACACGACGCTCTTCCGATCT [16-bp cell barcode] [10-bp UMI] TTTCTTATATGGG XXXXXXXXXXXXXXXXXXXXXXB(A)30------->
5'- CTACACGACGCTCTTCCGATCT --------> |--5'-CTACACGACGCTCTTCCGATCT [16-bp cell barcode] [10-bp UMI] TTTCTTATATGGG XXXXXXXXX...XXXXXXXXXB(pA)GTACTCTGCGTTGATACCACTGCTT -3' 3'-GATGTGCTGCGAGAAGGCTAGA [16-bp cell barcode] [10-bp UMI] AAAGAATATACCC XXXXXXXXX...XXXXXXXXXV(dT)CATGAGACGCAACTATGGTGACGAA -5' <--------GACGCAACTATGGTGACGAA -5'
Product 1 (TSO, cell barcode, UMI plus 5'-end of cDNA): 5'- CTACACGACGCTCTTCCGATCT [16-bp cell barcode] [10-bp UMI] TTTCTTATATGGG XXXXXXXXX...XXXXXXXXX*A -3' 3'- A*GATGTGCTGCGAGAAGGCTAGA [16-bp cell barcode] [10-bp UMI] AAAGAATATACCC XXXXXXXXX...XXXXXXXXX -5' Product 2 (middle of cDNA): 5'- XXXXXXXXXXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXXXXXXXXXX*A -3' 3'- A*XXXXXXXXXXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXXXXXXXXXX -5' Product 3 (3' of cDNA plus Poly-dT RT sequence): 5'-AAGCAGTGGTATCAACGCAGAGTAC (dT)VXXXXXXXXX...XXXXXXXXX*A -3' 3'- A*TTCGTCACCATAGTTGCGTCTCATG (pA)BXXXXXXXXX...XXXXXXXXX -5'
Product 1 (I assume the 5' end of TSO is blocked, so the adapter can only be ligated to the cDNA end. This is the only ampliable fragment: 5'- CTACACGACGCTCTTCCGATCT [16-bp cell barcode] [10-bp UMI] TTTCTTATATGGG XXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3' 3'- A*GATGTGCTGCGAGAAGGCTAGA [16-bp cell barcode] [10-bp UMI] AAAGAATATACCC XXX...XXXTCTAGCCTTCTCG -5' Product 2 (will not amplify efficiently due to semi-suppressive PCR??? not really sure about this): 5'-GCTCTTCCGATCT XXXXXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3' 3'-CACTGACCTCAAGTCTGCACACGAGAAGGCTAG AXXXXXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXXXXXTCTAGCCTTCTCG -5' Product 3 (I assume the 5' end of Poly-dT RT primer is blocked, so the adapter can only be ligated to the cDNA end. This produce is not amplifiable due to the use of the specific primers for amplification, see the next step): 5'-AAGCAGTGGTATCAACGCAGAGTAC (dT)VXXXXXXXXX...XXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3' 3'- A*TTCGTCACCATAGTTGCGTCTCATG (pA)BXXXXXXXXX...XXXXXXXXXTCTAGCCTTCTCG -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTC ---------> 5'-CTACACGACGCTCTTCCGATCT [16-bp cell barcode] [10-bp UMI] TTTCTTATATGGG XXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3' 3'- A*GATGTGCTGCGAGAAGGCTAGA [16-bp cell barcode] [10-bp UMI] AAAGAATATACCC XXX...XXXTCTAGCCTTCTCG -5' <-----------TGTGCAGACTTGAGGTCAGTG [8-bp sample index]TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNNNNNNNNNNN NNNNNNNNNN TTTCTTATATGGG XXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGATCGGAAGAGCGTCGTGTAGGGAAAGA NNNNNNNNNNNNNNNN NNNNNNNNNN AAAGAATATACCC XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 Truseq Read 1 16 bp 10 bp cDNATruseq Read 2 8 bpIllumina P7 cell barcode UMI Sample Index
5'- ACAC TCTTTCCCTACACGACGCTCTTCCGATCT ------------------------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGATCGGAAGAGCGTCGTGTAGGGAAAGA NNNNNNNNNNNNNNNN NNNNNNNNNN AAAGAATATACCC XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGATCGGAAGAGCGTCGTGTAGGGAAAGA NNNNNNNNNNNNNNNN NNNNNNNNNN AAAGAATATACCC XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNNNNNNNNNNN NNNNNNNNNN TTTCTTATATGGG XXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <-----TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'