Chromium Single Cell 5′ Gene Expression

Created

January 1, 2026

Modified

March 28, 2026

The 10x Chromium Single Cell 5′ Gene Expression kit captures the 5′ end of transcripts inside GEMs. Instead of barcoded oligo-dT primers on the beads (as in the 3′ kit), the 5′ kit uses a universal poly-dT RT primer for reverse transcription and places the barcoded TSO on the gel bead. The TSO anneals to non-templated Cs added by MMLV and carries the cell barcode and UMI. This 5′ orientation makes it ideal for paired V(D)J immune repertoire profiling.

Version Cell Barcode UMI Status
V1 (PN-220112) 16 bp 10 bp Discontinued
V2 (PN-1000264) 16 bp 10 bp Current
V3 (PN-2001129, GEM-X) 16 bp 12 bp Current

Library structure is identical between V1 and V2; V3 differs only in UMI length. The V1/V2 structure is shown below unless noted.

Adapter and Primer Sequences

Bead-TSO (V1 & V2):

|--5'- CTACACGACGCTCTTCCGATCT[16-bp CBC][10-bp UMI]TTTCTTATATrGrGrG -3'
       (rG = riboguanosine)

Bead-TSO (V3):

|--5'- CTACACGACGCTCTTCCGATCT[16-bp CBC][12-bp UMI]TTTCTTATATrGrGrG -3'

Poly-dT RT primer (PN-2000007):

5'- AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN -3'

cDNA Forward primer: 5'- CTACACGACGCTCTTCCGATCT -3'

cDNA Reverse primer: 5'- AAGCAGTGGTATCAACGCAG -3'

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

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

TruSeq adapter (dsDNA, T overhang, PN-220026):

5'-  GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
3'- TCTAGCCTTCTCG -5'

Library PCR primer 1 (PN-220111): 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC -3'

Library PCR primer 2 (PN-220103): 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp sample index]GTGACTGGAGTTCAGACGTGT -3'

Sample index sequencing primer: 5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'

Illumina P5: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Step-by-Step Library Generation

Step 1 — Reverse transcription with poly-dT RT primer

The poly-dT RT primer anneals to the poly-A tail of mRNA in solution. MMLV extends from the poly-A tail toward the 5′ cap.

                   <--------NV(T)30CATGAGACGCAACTATGGTGACGAA -5'  (poly-dT RT primer)
5'- mRNA-B(A)30

Step 2 — Terminal transferase adds non-templated Cs

MMLV’s terminal transferase activity adds 2–3 cytosines to the 3′ end of the nascent cDNA (which corresponds to the mRNA 5′ cap region).

    CCC-cDNA-NV(T)30CATGAGACGCAACTATGGTGACGAA -5'
5'- mRNA-B(A)30

Step 3 — cDNA capture by bead-TSO template switching

The non-templated Cs anneal to the riboG residues at the 3′ end of the bead-bound TSO. MMLV switches template and extends through the TSO, incorporating the CBC and UMI.

                                          <---------CCC-cDNA-NV(T)30CATGAGACGCAACTATGGTGACGAA -5'
|--5'- CTACACGACGCTCTTCCGATCT[CBC][UMI]TTTCTTATATGGG-cDNA-(pA)B-mRNA -3'

After extension:

|--5'- CTACACGACGCTCTTCCGATCT[CBC][UMI]TTTCTTATATGGG-cDNA-(pA)BGTACTCTGCGTTGATACCACTGCTT -3'
   3'- GATGTGCTGCGAGAAGGCTAGA[CBC][UMI]AAAGAATATACCC-cDNA-(dT)VNCATGAGACGCAACTATGGTGACGAA -5'

Step 4 — cDNA amplification

Full-length cDNA is amplified using the cDNA Forward primer (TruSeq Read 1 portion) and cDNA Reverse primer (poly-dT RT primer portion).

   5'- CTACACGACGCTCTTCCGATCT-------->
|--5'- CTACACGACGCTCTTCCGATCT[CBC][UMI]TTTCTTATATGGG-cDNA-(pA)BGTACTCTGCGTTGATACCACTGCTT -3'
   3'- GATGTGCTGCGAGAAGGCTAGA[CBC][UMI]AAAGAATATACCC-cDNA-(dT)VNCATGAGACGCAACTATGGTGACGAA -5'
                                                            <--------GACGCAACTATGGTGACGAA -5'

Step 5 — Fragmentase fragmentation and A-tailing

Amplified cDNA is enzymatically fragmented and A-tailed, generating three fragment classes:

Product 1 (TSO + CBC + UMI + 5′ cDNA — the only amplifiable fragment):

5'-   CTACACGACGCTCTTCCGATCT[CBC][UMI]TTTCTTATATGGG-cDNA*A -3'
3'- A*GATGTGCTGCGAGAAGGCTAGA[CBC][UMI]AAAGAATATACCC-cDNA    -5'

Product 2 (internal — semi-suppressive PCR, not amplifiable):

5'-   cDNA*A -3'
3'- A* cDNA  -5'

Product 3 (3′ poly-dT RT primer end — 5′ end blocked, not amplifiable):

5'-   AAGCAGTGGTATCAACGCAGAGTAC(dT)VN-cDNA*A -3'
3'- A*TTCGTCACCATAGTTGCGTCTCATG(pA)B-cDNA    -5'

Step 6 — TruSeq adapter ligation

TruSeq adapter is ligated to the A-tailed Product 1:

5'-   CTACACGACGCTCTTCCGATCT[CBC][UMI]TTTCTTATATGGG-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
3'- A*GATGTGCTGCGAGAAGGCTAGA[CBC][UMI]AAAGAATATACCC-cDNA-TCTAGCCTTCTCG -5'

Step 7 — Library PCR amplification

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-------->
                      5'-   CTACACGACGCTCTTCCGATCT[CBC][UMI]TTTCTTATATGGG-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
                      3'- A*GATGTGCTGCGAGAAGGCTAGA[CBC][UMI]AAAGAATATACCC-cDNA-TCTAGCCTTCTCG                         -5'
                                                                      <---------TGTGCAGACTTGAGGTCAGTG[8-bp idx]TAGAGCATACGGCAGAAGACGAAC -5'

Final Library Structure

V1 and V2 (16 bp CBC + 10 bp UMI)

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGG-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCC-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5                   TruSeq Read 1                16 bp CBC     10 bp UMI    TSO        cDNA              TruSeq Read 2              8 bp idx       Illumina P7

V3 (16 bp CBC + 12 bp UMI)

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGG-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCC-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5                   TruSeq Read 1                16 bp CBC     12 bp UMI    TSO        cDNA              TruSeq Read 2              8 bp idx       Illumina P7

Library Sequencing

Step 1 — Read 1: cell barcode + UMI (bottom strand as template)

                         5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCC-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

26 cycles (V1/V2) or 28 cycles (V3).

Step 2 — Index 1 (i7): sample index (bottom strand as template)

                                                                                           5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCC-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

8 cycles.

Step 3 — Read 2: cDNA (top strand as template, cluster regeneration)

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGG-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                              <------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'

98 cycles (cDNA).

Key Points

  • Read 1 sequences the cell barcode and UMI — no cDNA content.
  • Read 2 sequences cDNA from the 5′ end of transcripts — used for gene alignment.
  • The TSO sequence (TTTCTTATATGGG) appears at the junction between the UMI and cDNA; Cell Ranger trims this during preprocessing.
  • Only i7 sample index is used.
  • This assay is often run with V(D)J enrichment primers to simultaneously profile TCR/BCR sequences.
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