Visium HD 3′ Gene Expression

Created

January 1, 2026

Modified

March 28, 2026

Visium HD 3′ is the whole-transcriptome, probe-free counterpart to the Visium HD WT Panel. It uses the same 2 µm bin continuous-lawn slide architecture but captures polyadenylated mRNA directly via poly-T oligonucleotides — no probe pairs or ligation step required. This enables de novo discovery across the entire transcriptome, including non-coding RNAs, and extends compatibility to non-human species beyond those covered by the WT Panel probe design.

Feature Visium HD WT Panel Visium HD 3′
Capture method Probe pairs (WT Panel) poly-T (whole transcriptome)
Gene coverage ~18,000 genes (curated panel) Whole transcriptome (de novo)
Non-human species Human / mouse panels only Broad species compatibility
CytAssist Required Required
Resolution 2 µm bins 2 µm bins
Sample compatibility FFPE, FF, Fixed-frozen FFPE, FF, Fixed-frozen

Like Visium v1, the 3′ assay uses a TSO-based template switching mechanism; unlike v1 it operates at 2 µm resolution and requires CytAssist for tissue transfer.

Capture Bin Oligonucleotide

5'- CTACACGACGCTCTTCCGATCT[16-bp spatial BC][12-bp UMI](T)30VN -3'    (surface-bound, 2 µm bin)

Adapter and Primer Sequences

Capture bin oligo:

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 preparation and CytAssist transfer

Tissue section (5–10 µm) is placed on a standard glass slide, fixed (if FFPE), and prepared histologically. After staining and imaging, the section is permeabilised to release mRNA. The CytAssist instrument transfers released mRNA from the glass slide to the Visium HD 3′ capture area.

Step 2 — mRNA capture by poly-T bin oligos

Poly-A mRNA binds to the poly-T capture oligos on the 2 µm bin surface:

(tissue section)
      mRNA  5'--XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX(A)n -3'
                                                   |||
slide 5'- CTACACGACGCTCTTCCGATCT[16-bp spatial BC][12-bp UMI](T)30VN -3'  (2 µm bin oligo)

Step 3 — In situ reverse transcription on capture area

MMLV reverse transcriptase extends from the capture oligo primer through the mRNA template:

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

MMLV’s terminal transferase activity adds non-templated CCC at the 3′ end.

Step 4 — TSO template switching

The TSO riboguanosines (rGrGrG) anneal to the non-templated CCC; MMLV switches template and extends through the TSO:

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

Step 5 — cDNA release and amplification

Spatial barcoded cDNA is released from the slide and PCR amplified:

   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 6 — Fragmentase fragmentation and A-tailing

Three fragment classes are generated:

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

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

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

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

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

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

Step 7 — TruSeq adapter ligation

TruSeq adapter is 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 8 — 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 (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 (2 µm bin) + 12 bp UMI. No cDNA content.
  • Read 2 (98 cycles): cDNA sequence — aligned to the genome/transcriptome for gene quantification.
  • Unlike the HD WT Panel, there is no probe barcode in Read 2; gene identity comes from genome alignment (like bulk 3′ RNA-seq).
  • CytAssist is required to transfer captured mRNA from the glass slide permeabilisation surface to the Visium HD slide.
  • Space Ranger HD processes the data: maps Read 2 to the genome, bins reads by spatial barcode, and builds the multi-resolution matrix.
  • Expanded species compatibility compared to the WT Panel, since no species-specific probes are required.
  • Compatible with FFPE, fresh-frozen, and fixed-frozen tissues when used with the appropriate tissue preparation protocol.
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