Chromium Single Cell 3′ Gene Expression

The 10x Chromium Single Cell 3′ Gene Expression kit captures polyadenylated mRNA at the 3′ end inside nano-litre droplets (GEMs). Each GEM contains a single cell and one gel bead. The gel bead carries tens of thousands of barcoded oligo-dT primers, one unique 16-bp cell barcode (CBC) and many unique UMI tags.

Multiple chemistry versions exist:

Version	Cell Barcode	UMI	Status
V2	16 bp	10 bp	Discontinued
V3 / V3.1	16 bp	12 bp	Current
V4 (GEM-X)	16 bp	12 bp	Current

Library structure is identical between V3, V3.1, and V4; V2 differs only in UMI length. Unless noted, V2 steps are drawn below.

Adapter and Primer Sequences

Bead oligo-dT (V2):

|--5'- CTACACGACGCTCTTCCGATCT[16-bp CBC][10-bp UMI](T)30VN -3'

Bead oligo-dT (V3 / V3.1 / V4):

|--5'- CTACACGACGCTCTTCCGATCT[16-bp CBC][12-bp UMI](T)30VN -3'

Template Switching Oligo (TSO):

5'- AAGCAGTGGTATCAACGCAGAGTACATGGGRGG -3'
    (rG = riboguanosine)

cDNA Forward primer: 5'- CTACACGACGCTCTTCCGATCT -3'

cDNA Reverse primer:

V2:       5'- AAGCAGTGGTATCAACGCAGAGTACAT -3'
V3/V3.1/V4: 5'- AAGCAGTGGTATCAACGCAGAG -3'

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

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

TruSeq adapter (dsDNA, T overhang):

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

Library PCR primer 1: 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC -3'

Library PCR primer 2: 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 — mRNA capture and reverse transcription inside GEMs

Poly-A mRNA is captured by the oligo-dT bead primer inside the droplet. MMLV reverse transcriptase extends using the mRNA as template.

|--5'- CTACACGACGCTCTTCCGATCT[16-bp CBC][10-bp UMI](T)30VN-------->
                                                              (A)30B-mRNA-5'

Step 2 — Terminal transferase adds extra Cs (non-templated)

MMLV’s terminal transferase activity appends 2–3 extra cytosines to the 3′ end of the cDNA.

|--5'- CTACACGACGCTCTTCCGATCT[16-bp CBC][10-bp UMI](dT)VN-cDNA-CCC -3'
                              (pA)B-mRNA-5'

Step 3 — TSO template switching for second strand synthesis

The TSO’s 3′ riboguanosines anneal to the non-templated Cs, and MMLV switches template to extend through the TSO.

|--5'- CTACACGACGCTCTTCCGATCT[16-bp CBC][10-bp UMI](dT)VN-cDNA-CCC---------->
                                        <---------GGGrGrGrGATGTACTCTGCGTTGATACCACTGCTT -5'  (TSO)

Full-length first-strand cDNA:

|--5'- CTACACGACGCTCTTCCGATCT[CBC][UMI](dT)VN-cDNA-CCATGTACTCTGCGTTGATACCACTGCTT -3'
   3'- GATGTGCTGCGAGAAGGCTAGA[CBC][UMI](pA)B-cDNA-GGTACATGAGACGCAACTATGGTGACGAA -5'

Step 4 — cDNA amplification with Forward and Reverse primers

PCR amplifies full-length cDNA using the cDNA Forward primer (anneals to TruSeq Read 1 portion) and cDNA Reverse primer (anneals to TSO portion).

   5'- CTACACGACGCTCTTCCGATCT-------->
|--5'- CTACACGACGCTCTTCCGATCT[CBC][UMI](dT)VN-cDNA-CCATGTACTCTGCGTTGATACCACTGCTT -3'
   3'- GATGTGCTGCGAGAAGGCTAGA[CBC][UMI](pA)B-cDNA-GGTACATGAGACGCAACTATGGTGACGAA -5'
                                                             <--------TACATGAGACGCAACTATGGTGACGAA -5'

Step 5 — Fragmentase fragmentation and A-tailing

The amplified cDNA is enzymatically fragmented and A-tailed, producing three fragment classes:

Product 1 (TSO-containing 5′ fragment — not amplifiable in next step):

5'-   AAGCAGTGGTATCAACGCAGAGTACATGGG-cDNA-*A -3'
3'- A*TTCGTCACCATAGTTGCGTCTCATGTACCC-cDNA    -5'

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

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

Product 3 (Read 1 + CBC + UMI + 3′ cDNA — the only amplifiable fragment):

5'-   CTACACGACGCTCTTCCGATCT[CBC][UMI](dT)VN-cDNA*A -3'
3'- A*GATGTGCTGCGAGAAGGCTAGA[CBC][UMI](pA)B-cDNA    -5'

Step 6 — TruSeq adapter ligation

Double-stranded TruSeq adapter (T overhang) is ligated to the A-tailed Product 3:

5'-   CTACACGACGCTCTTCCGATCT[CBC][UMI](dT)VN-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
3'- A*GATGTGCTGCGAGAAGGCTAGA[CBC][UMI](pA)B-cDNA-TCTAGCCTTCTCG -5'

Step 7 — Library PCR amplification

Library PCR Primer 1 (extends from P5 through TruSeq Read 1) and Library PCR Primer 2 (carries sample index and P7) amplify the final library.

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-------->
                      5'-   CTACACGACGCTCTTCCGATCT[CBC][UMI](dT)VN-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
                      3'- A*GATGTGCTGCGAGAAGGCTAGA[CBC][UMI](pA)B-cDNA-TCTAGCCTTCTCG                         -5'
                                                               <---------TGTGCAGACTTGAGGTCAGTG[8-bp idx]TAGAGCATACGGCAGAAGACGAAC -5'

Final Library Structure

V2 (16 bp CBC + 10 bp UMI)

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNN(dT)VN-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNN(pA)B-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5                   TruSeq Read 1                16 bp CBC     10 bp UMI          cDNA              TruSeq Read 2              8 bp idx       Illumina P7

V3 / V3.1 / V4 (16 bp CBC + 12 bp UMI)

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNNN(dT)VN-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNNN(pA)B-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5                   TruSeq Read 1                16 bp CBC     12 bp UMI          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'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNN(pA)B-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

Sequence 26 cycles (V2: 16 bp CBC + 10 bp UMI) or 28 cycles (V3+: 16 bp CBC + 12 bp UMI).

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

                                                                               5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNN(pA)B-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

8 cycles.

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

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNN(dT)VN-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                          <------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'

98 cycles (cDNA).

Key Points

Read 1 carries the cell barcode and UMI — it does not contain cDNA sequence.
Read 2 carries the cDNA fragment — used for gene alignment.
Only one sample index (i7) is used; i5 is not used in this assay.
Cell Ranger uses the whitelist for CBC correction during demultiplexing.

--- title: "Chromium Single Cell 3′ Gene Expression" date: 2026-01-01 published-title: Created date-modified: last-modified title-block-banner: "#212529" toc: true toc-location: left toc-title: "Contents" execute: eval: false format: html: code-tools: source: true toggle: true css: 10x.css --- The 10x Chromium Single Cell 3′ Gene Expression kit captures polyadenylated mRNA at the 3′ end inside nano-litre droplets (GEMs). Each GEM contains a single cell and one gel bead. The gel bead carries tens of thousands of barcoded oligo-dT primers, one unique 16-bp **cell barcode (CBC)** and many unique **UMI** tags. Multiple chemistry versions exist: | Version | Cell Barcode | UMI | Status | |---------|-------------|-----|--------| | V2 | 16 bp | 10 bp | Discontinued | | V3 / V3.1 | 16 bp | 12 bp | Current | | V4 (GEM-X) | 16 bp | 12 bp | Current | Library structure is identical between V3, V3.1, and V4; V2 differs only in UMI length. Unless noted, V2 steps are drawn below. --- ## Adapter and Primer Sequences **Bead oligo-dT (V2):** ``` |--5'- CTACACGACGCTCTTCCGATCT[16-bp CBC][10-bp UMI](T)30VN -3' ``` **Bead oligo-dT (V3 / V3.1 / V4):** ``` |--5'- CTACACGACGCTCTTCCGATCT[16-bp CBC][12-bp UMI](T)30VN -3' ``` **Template Switching Oligo (TSO):** ``` 5'- AAGCAGTGGTATCAACGCAGAGTACATGGGRGG -3' (rG = riboguanosine) ``` **cDNA Forward primer:** `5'- CTACACGACGCTCTTCCGATCT -3'` **cDNA Reverse primer:** ``` V2: 5'- AAGCAGTGGTATCAACGCAGAGTACAT -3' V3/V3.1/V4: 5'- AAGCAGTGGTATCAACGCAGAG -3' ``` **TruSeq Read 1 primer:** `5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT -3'` **TruSeq Read 2 primer:** `5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3'` **TruSeq adapter (dsDNA, T overhang):** ``` 5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3' 3'- TCTAGCCTTCTCG -5' ``` **Library PCR primer 1:** `5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC -3'` **Library PCR primer 2:** `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 — mRNA capture and reverse transcription inside GEMs Poly-A mRNA is captured by the oligo-dT bead primer inside the droplet. MMLV reverse transcriptase extends using the mRNA as template. <pre class="seq"> |--5'- <s5>CTACACGACGCTCTTCCGATCT</s5><cbc>[16-bp CBC]</cbc><umi>[10-bp UMI]</umi>(T)30VN--------> (A)30B-mRNA-5' </pre> ### Step 2 — Terminal transferase adds extra Cs (non-templated) MMLV's terminal transferase activity appends 2–3 extra cytosines to the 3′ end of the cDNA. <pre class="seq"> |--5'- <s5>CTACACGACGCTCTTCCGATCT</s5><cbc>[16-bp CBC]</cbc><umi>[10-bp UMI]</umi>(dT)VN-cDNA-CCC -3' (pA)B-mRNA-5' </pre> ### Step 3 — TSO template switching for second strand synthesis The TSO's 3′ riboguanosines anneal to the non-templated Cs, and MMLV switches template to extend through the TSO. <pre class="seq"> |--5'- <s5>CTACACGACGCTCTTCCGATCT</s5><cbc>[16-bp CBC]</cbc><umi>[10-bp UMI]</umi>(dT)VN-cDNA-CCC----------> <---------GGGrGrGrGATGTACTCTGCGTTGATACCACTGCTT -5' (TSO) </pre> Full-length first-strand cDNA: <pre class="seq"> |--5'- <s5>CTACACGACGCTCTTCCGATCT</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(dT)VN-cDNA-<tso>CCATGTACTCTGCGTTGATACCACTGCTT</tso> -3' 3'- <s5>GATGTGCTGCGAGAAGGCTAGA</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(pA)B-cDNA-<tso>GGTACATGAGACGCAACTATGGTGACGAA</tso> -5' </pre> ### Step 4 — cDNA amplification with Forward and Reverse primers PCR amplifies full-length cDNA using the cDNA Forward primer (anneals to TruSeq Read 1 portion) and cDNA Reverse primer (anneals to TSO portion). <pre class="seq"> 5'- <s5>CTACACGACGCTCTTCCGATCT</s5>--------> |--5'- <s5>CTACACGACGCTCTTCCGATCT</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(dT)VN-cDNA-<tso>CCATGTACTCTGCGTTGATACCACTGCTT</tso> -3' 3'- <s5>GATGTGCTGCGAGAAGGCTAGA</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(pA)B-cDNA-<tso>GGTACATGAGACGCAACTATGGTGACGAA</tso> -5' <--------<tso>TACATGAGACGCAACTATGGTGACGAA</tso> -5' </pre> ### Step 5 — Fragmentase fragmentation and A-tailing The amplified cDNA is enzymatically fragmented and A-tailed, producing three fragment classes: **Product 1** (TSO-containing 5′ fragment — not amplifiable in next step): <pre class="seq"> 5'- <tso>AAGCAGTGGTATCAACGCAGAGTACATGGG</tso>-cDNA-*A -3' 3'- A*<tso>TTCGTCACCATAGTTGCGTCTCATGTACCC</tso>-cDNA -5' </pre> **Product 2** (internal fragment — semi-suppressive PCR, not amplifiable): <pre class="seq"> 5'- cDNA ...*A -3' 3'- A* cDNA -5' </pre> **Product 3** (Read 1 + CBC + UMI + 3′ cDNA — the **only amplifiable fragment**): <pre class="seq"> 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(dT)VN-cDNA*A -3' 3'- A*<s5>GATGTGCTGCGAGAAGGCTAGA</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(pA)B-cDNA -5' </pre> ### Step 6 — TruSeq adapter ligation Double-stranded TruSeq adapter (T overhang) is ligated to the A-tailed Product 3: <pre class="seq"> 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(dT)VN-cDNA-<s7>AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC</s7> -3' 3'- A*<s5>GATGTGCTGCGAGAAGGCTAGA</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(pA)B-cDNA-<s7>TCTAGCCTTCTCG</s7> -5' </pre> ### Step 7 — Library PCR amplification Library PCR Primer 1 (extends from P5 through TruSeq Read 1) and Library PCR Primer 2 (carries sample index and P7) amplify the final library. <pre class="seq"> 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5><s5>TCTTTCCCTACACGACGCTC</s5>--------> 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(dT)VN-cDNA-<s7>AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC</s7> -3' 3'- A*<s5>GATGTGCTGCGAGAAGGCTAGA</s5><cbc>[CBC]</cbc><umi>[UMI]</umi>(pA)B-cDNA-<s7>TCTAGCCTTCTCG</s7> -5' <---------<s7>TGTGCAGACTTGAGGTCAGTG</s7><t7>[8-bp idx]</t7><p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5' </pre> --- ## Final Library Structure ### V2 (16 bp CBC + 10 bp UMI) <pre class="seq"> 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5><s5>TCTTTCCCTACACGACGCTCTTCCGATCT</s5><cbc>NNNNNNNNNNNNNNNN</cbc><umi>NNNNNNNNNN</umi>(dT)VN-cDNA-<s7>AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC</s7><t7>NNNNNNNN</t7><p7>ATCTCGTATGCCGTCTTCTGCTTG</p7> -3' 3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5><s5>AGAAAGGGATGTGCTGCGAGAAGGCTAGA</s5><cbc>NNNNNNNNNNNNNNNN</cbc><umi>NNNNNNNNNN</umi>(pA)B-cDNA-<s7>TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG</s7><t7>NNNNNNNN</t7><p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5' <p5>Illumina P5</p5> <s5>TruSeq Read 1</s5> <cbc>16 bp CBC</cbc> <umi>10 bp UMI</umi> cDNA <s7>TruSeq Read 2</s7> <t7>8 bp idx</t7> <p7>Illumina P7</p7> </pre> ### V3 / V3.1 / V4 (16 bp CBC + 12 bp UMI) <pre class="seq"> 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5><s5>TCTTTCCCTACACGACGCTCTTCCGATCT</s5><cbc>NNNNNNNNNNNNNNNN</cbc><umi>NNNNNNNNNNNN</umi>(dT)VN-cDNA-<s7>AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC</s7><t7>NNNNNNNN</t7><p7>ATCTCGTATGCCGTCTTCTGCTTG</p7> -3' 3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5><s5>AGAAAGGGATGTGCTGCGAGAAGGCTAGA</s5><cbc>NNNNNNNNNNNNNNNN</cbc><umi>NNNNNNNNNNNN</umi>(pA)B-cDNA-<s7>TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG</s7><t7>NNNNNNNN</t7><p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5' <p5>Illumina P5</p5> <s5>TruSeq Read 1</s5> <cbc>16 bp CBC</cbc> <umi>12 bp UMI</umi> cDNA <s7>TruSeq Read 2</s7> <t7>8 bp idx</t7> <p7>Illumina P7</p7> </pre> --- ## Library Sequencing ### Step 1 — Read 1: cell barcode + UMI (bottom strand as template) <pre class="seq"> 5'- <s5>ACACTCTTTCCCTACACGACGCTCTTCCGATCT</s5>-------------------------> 3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5><s5>AGAAAGGGATGTGCTGCGAGAAGGCTAGA</s5><cbc>NNNNNNNNNNNNNNNN</cbc><umi>NNNNNNNNNN</umi>(pA)B-cDNA-<s7>TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG</s7><t7>NNNNNNNN</t7><p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5' </pre> Sequence 26 cycles (V2: 16 bp CBC + 10 bp UMI) or 28 cycles (V3+: 16 bp CBC + 12 bp UMI). ### Step 2 — Index 1 (i7): sample index (bottom strand as template) <pre class="seq"> 5'- <s7>GATCGGAAGAGCACACGTCTGAACTCCAGTCAC</s7>-------> 3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5><s5>AGAAAGGGATGTGCTGCGAGAAGGCTAGA</s5><cbc>NNNNNNNNNNNNNNNN</cbc><umi>NNNNNNNNNN</umi>(pA)B-cDNA-<s7>TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG</s7><t7>NNNNNNNN</t7><p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5' </pre> 8 cycles. ### Step 3 — Read 2: cDNA (top strand as template, cluster regeneration) <pre class="seq"> 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5><s5>TCTTTCCCTACACGACGCTCTTCCGATCT</s5><cbc>NNNNNNNNNNNNNNNN</cbc><umi>NNNNNNNNNN</umi>(dT)VN-cDNA-<s7>AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC</s7><t7>NNNNNNNN</t7><p7>ATCTCGTATGCCGTCTTCTGCTTG</p7> -3' <------<s7>TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG</s7> -5' </pre> 98 cycles (cDNA). --- ## Key Points - **Read 1** carries the cell barcode and UMI — it does **not** contain cDNA sequence. - **Read 2** carries the cDNA fragment — used for gene alignment. - Only one sample index (i7) is used; i5 is not used in this assay. - Cell Ranger uses the whitelist for CBC correction during demultiplexing.