Visium Spatial Gene Expression v1

Visium v1 is 10x Genomics’ first-generation spatial gene expression technology. It captures polyadenylated mRNA directly from a fresh-frozen (FF) tissue section placed on a patterned slide. Each slide contains 4 capture areas, each with ~5,000 barcoded capture spots arranged in a hexagonal array. Spots are 55 µm in diameter with 100 µm centre-to-centre spacing.

Each capture spot contains millions of poly-T oligonucleotides covalently attached to the slide surface. Each oligo consists of:

A partial TruSeq Read 1 sequence (for library construction)
A 16-bp spatial barcode (sbc) uniquely encoding the X-Y position of the spot
A 12-bp UMI
A poly-T tail (VN anchor + 30 Ts) for mRNA capture

The library is then constructed and sequenced using standard TruSeq chemistry.

Capture Spot Oligonucleotide

5'- CTACACGACGCTCTTCCGATCT[16-bp spatial BC][12-bp UMI](T)30VN -3'    (surface-bound)

The spatial barcode encodes the row and column identity of each spot. A whitelist of all valid 16 bp spatial barcodes is used by Space Ranger for spot assignment.

Adapter and Primer Sequences

Capture spot oligo on slide:

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

Template Switching Oligo (TSO):

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

cDNA amplification primer: 5'- CTACACGACGCTCTTCCGATCT -3'

cDNA Reverse primer: 5'- AAGCAGTGGTATCAACGCAGAGTACAT -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'

Illumina P5: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Step-by-Step Library Generation

Step 1 — Tissue placement and mRNA capture

Fresh-frozen tissue section (10 µm) is placed on the Visium capture area. After H&E staining and imaging, the section is permeabilised. Released mRNA diffuses down and is captured by the poly-T oligos at the spots beneath the tissue.

(tissue above slide)
      mRNA  5'--XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX(A)n -3'
                                                   |||
slide 5'- CTACACGACGCTCTTCCGATCT[16-bp spatial BC][12-bp UMI](T)30VN -3'  (surface-bound)

Step 2 — In situ reverse transcription

Reverse transcriptase (MMLV) extends the captured mRNA using the surface-bound oligo as primer:

slide 5'- CTACACGACGCTCTTCCGATCT[spatial BC][UMI](dT)VN-cDNA-CCC -3'
                                                          (pA)-mRNA-5'

MMLV adds non-templated Cs at the 3′ end of the cDNA.

Step 3 — TSO template switching

The TSO riboguanosines anneal to the non-templated Cs; MMLV extends through the TSO sequence:

slide 5'- CTACACGACGCTCTTCCGATCT[spatial BC][UMI](dT)VN-cDNA-CCATGTACTCTGCGTTGATACCACTGCTT -3'
      3'-                                                              GGTACATGAGACGCAACTATGGTGACGAA -5'  (TSO)

Step 4 — cDNA release and amplification

cDNA is released from the slide and amplified by PCR using the cDNA amplification primer (anneals to TruSeq Read 1 portion) and cDNA Reverse primer (anneals to TSO portion).

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

Step 5 — Fragmentase fragmentation and A-tailing

Amplified cDNA is enzymatically fragmented and A-tailed:

Product 1 (TSO 5′ fragment — not amplifiable):

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

Product 2 (internal — not amplifiable):

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

Product 3 (TruSeq Read 1 + spatial BC + UMI + 3′ cDNA — only amplifiable product):

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

Step 6 — TruSeq adapter ligation

TruSeq adapter ligated to Product 3:

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

Step 7 — Library PCR amplification

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

Final Library Structure

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNNN(dT)VN-cDNA-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNNN(pA)B-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5                   TruSeq Read 1              16 bp spatial BC  12 bp UMI       cDNA              TruSeq Read 2              8 bp idx       Illumina P7

Library Sequencing

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

                         5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNNN(pA)B-cDNA-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

28 cycles (16 bp spatial BC + 12 bp UMI).

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

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

8 cycles.

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

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

98 cycles (cDNA).

Key Points

Read 1 (28 cycles): 16 bp spatial barcode + 12 bp UMI. No cDNA content.
Read 2 (98 cycles): cDNA fragment — used for transcript alignment.
The spatial barcode whitelist maps each 16 bp sequence to a known X-Y coordinate on the capture area.
Space Ranger processes the FASTQ files, aligns transcripts, and generates a spatial gene expression matrix.
Tissue placement and permeabilisation are critical steps — poor permeabilisation reduces RNA capture efficiency.
Compatible with fresh-frozen tissue only. For FFPE, use Visium v2 (WT Panel) chemistry.

--- title: "Visium Spatial Gene Expression v1" 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 --- Visium v1 is 10x Genomics' first-generation spatial gene expression technology. It captures polyadenylated mRNA directly from a fresh-frozen (FF) tissue section placed on a patterned slide. Each slide contains **4 capture areas**, each with ~**5,000 barcoded capture spots** arranged in a hexagonal array. Spots are 55 µm in diameter with 100 µm centre-to-centre spacing. Each capture spot contains millions of poly-T oligonucleotides covalently attached to the slide surface. Each oligo consists of: - A partial TruSeq Read 1 sequence (for library construction) - A **16-bp spatial barcode** (`sbc`) uniquely encoding the X-Y position of the spot - A **12-bp UMI** - A poly-T tail (VN anchor + 30 Ts) for mRNA capture The library is then constructed and sequenced using standard TruSeq chemistry. --- ## Capture Spot Oligonucleotide <pre class="seq"> 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><sbc>[16-bp spatial BC]</sbc><umi>[12-bp UMI]</umi>(T)30VN -3' (surface-bound) </pre> The spatial barcode encodes the row and column identity of each spot. A whitelist of all valid 16 bp spatial barcodes is used by Space Ranger for spot assignment. --- ## Adapter and Primer Sequences **Capture spot oligo on slide:** ``` 5'- CTACACGACGCTCTTCCGATCT[16-bp spatial BC][12-bp UMI](T)30VN -3' ``` **Template Switching Oligo (TSO)**: ``` 5'- AAGCAGTGGTATCAACGCAGAGTACATGGGRGG -3' (rG = riboguanosine) ``` **cDNA amplification primer:** `5'- CTACACGACGCTCTTCCGATCT -3'` **cDNA Reverse primer:** `5'- AAGCAGTGGTATCAACGCAGAGTACAT -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'` **Illumina P5:** `5'- AATGATACGGCGACCACCGAGATCTACAC -3'` **Illumina P7:** `5'- CAAGCAGAAGACGGCATACGAGAT -3'` --- ## Step-by-Step Library Generation ### Step 1 — Tissue placement and mRNA capture Fresh-frozen tissue section (10 µm) is placed on the Visium capture area. After H&E staining and imaging, the section is permeabilised. Released mRNA diffuses down and is captured by the poly-T oligos at the spots beneath the tissue. <pre class="seq"> (tissue above slide) mRNA 5'--XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX(A)n -3' ||| slide 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><sbc>[16-bp spatial BC]</sbc><umi>[12-bp UMI]</umi>(T)30VN -3' (surface-bound) </pre> ### Step 2 — In situ reverse transcription Reverse transcriptase (MMLV) extends the captured mRNA using the surface-bound oligo as primer: <pre class="seq"> slide 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><sbc>[spatial BC]</sbc><umi>[UMI]</umi>(dT)VN-cDNA-CCC -3' (pA)-mRNA-5' </pre> MMLV adds non-templated Cs at the 3′ end of the cDNA. ### Step 3 — TSO template switching The TSO riboguanosines anneal to the non-templated Cs; MMLV extends through the TSO sequence: <pre class="seq"> slide 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><sbc>[spatial BC]</sbc><umi>[UMI]</umi>(dT)VN-cDNA-<tso>CCATGTACTCTGCGTTGATACCACTGCTT</tso> -3' 3'- <tso>GGTACATGAGACGCAACTATGGTGACGAA</tso> -5' (TSO) </pre> ### Step 4 — cDNA release and amplification cDNA is released from the slide and amplified by PCR using the cDNA amplification 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><sbc>[spatial BC]</sbc><umi>[UMI]</umi>(dT)VN-cDNA-<tso>CCATGTACTCTGCGTTGATACCACTGCTT</tso> -3' 3'- <s5>GATGTGCTGCGAGAAGGCTAGA</s5><sbc>[spatial BC]</sbc><umi>[UMI]</umi>(pA)B-cDNA-<tso>GGTACATGAGACGCAACTATGGTGACGAA</tso> -5' <-------<tso>TACATGAGACGCAACTATGGTGACGAA</tso> -5' </pre> ### Step 5 — Fragmentase fragmentation and A-tailing Amplified cDNA is enzymatically fragmented and A-tailed: **Product 1** (TSO 5′ fragment — not amplifiable): <pre class="seq"> 5'- <tso>AAGCAGTGGTATCAACGCAGAGTACATGGG</tso>-cDNA*A -3' 3'- A*<tso>TTCGTCACCATAGTTGCGTCTCATGTACCC</tso>-cDNA -5' </pre> **Product 2** (internal — not amplifiable): <pre class="seq"> 5'- cDNA*A -3' 3'- A* cDNA -5' </pre> **Product 3** (TruSeq Read 1 + spatial BC + UMI + 3′ cDNA — **only amplifiable product**): <pre class="seq"> 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><sbc>[spatial BC]</sbc><umi>[UMI]</umi>(dT)VN-cDNA*A -3' 3'- A*<s5>GATGTGCTGCGAGAAGGCTAGA</s5><sbc>[spatial BC]</sbc><umi>[UMI]</umi>(pA)B-cDNA -5' </pre> ### Step 6 — TruSeq adapter ligation TruSeq adapter ligated to Product 3: <pre class="seq"> 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><sbc>[spatial BC]</sbc><umi>[UMI]</umi>(dT)VN-cDNA-<s7>AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC</s7> -3' 3'- A*<s5>GATGTGCTGCGAGAAGGCTAGA</s5><sbc>[spatial BC]</sbc><umi>[UMI]</umi>(pA)B-cDNA-<s7>TCTAGCCTTCTCG</s7> -5' </pre> ### Step 7 — Library PCR amplification <pre class="seq"> 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5><s5>TCTTTCCCTACACGACGCTC</s5>--------> 5'- <s5>CTACACGACGCTCTTCCGATCT</s5><sbc>[spatial BC]</sbc><umi>[UMI]</umi>(dT)VN-cDNA-<s7>AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC</s7> -3' 3'- <s5>GATGTGCTGCGAGAAGGCTAGA</s5><sbc>[spatial BC]</sbc><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 <pre class="seq"> 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5><s5>TCTTTCCCTACACGACGCTCTTCCGATCT</s5><sbc>NNNNNNNNNNNNNNNN</sbc><umi>NNNNNNNNNNNN</umi>(dT)VN-cDNA-<s7>AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC</s7><t7>NNNNNNNN</t7><p7>ATCTCGTATGCCGTCTTCTGCTTG</p7> -3' 3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5><s5>AGAAAGGGATGTGCTGCGAGAAGGCTAGA</s5><sbc>NNNNNNNNNNNNNNNN</sbc><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> <sbc>16 bp spatial BC</sbc> <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: spatial barcode + UMI (bottom strand as template) <pre class="seq"> 5'- <s5>ACACTCTTTCCCTACACGACGCTCTTCCGATCT</s5>-------------------------> 3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5><s5>AGAAAGGGATGTGCTGCGAGAAGGCTAGA</s5><sbc>NNNNNNNNNNNNNNNN</sbc><umi>NNNNNNNNNNNN</umi>(pA)B-cDNA-<s7>TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG</s7><t7>NNNNNNNN</t7><p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5' </pre> 28 cycles (16 bp spatial BC + 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><sbc>NNNNNNNNNNNNNNNN</sbc><umi>NNNNNNNNNNNN</umi>(pA)B-cDNA-<s7>TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG</s7><t7>NNNNNNNN</t7><p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5' </pre> 8 cycles. ### Step 3 — Read 2: cDNA sequence (top strand as template, cluster regeneration) <pre class="seq"> 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5><s5>TCTTTCCCTACACGACGCTCTTCCGATCT</s5><sbc>NNNNNNNNNNNNNNNN</sbc><umi>NNNNNNNNNNNN</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** (28 cycles): 16 bp spatial barcode + 12 bp UMI. No cDNA content. - **Read 2** (98 cycles): cDNA fragment — used for transcript alignment. - The spatial barcode whitelist maps each 16 bp sequence to a known X-Y coordinate on the capture area. - Space Ranger processes the FASTQ files, aligns transcripts, and generates a spatial gene expression matrix. - Tissue placement and permeabilisation are critical steps — poor permeabilisation reduces RNA capture efficiency. - Compatible with **fresh-frozen tissue only**. For FFPE, use Visium v2 (WT Panel) chemistry.