**Biomaterials**

Human colon cancer tissue was obtained from BioIVT.

**Sample preparation**

A 10 µm section was taken with a cryostat (Epredia CryoStar NX70). Sectioning, deparaffinization, H&E staining and imaging followed the Visium HD Fresh Frozen Tissue Preparation Handbook 2.0 ([CG001677](/support/spatial-gene-expression-hd/documentation/steps/tissue-prep-for-fresh-frozen/visium-hd-ff-tissue-preparation-handbook-2-0)).

**Imaging**

- Image type: H&E
- Microscope: Olympus VS200 Slide Scanner
- Objective magnification: 20X
- Numerical Aperture: 0.8
- ScopeLED light source: VS200 LED, Olympus integrated bright field source
- Camera: iDS VS-264C, Olympus scanner integrated camera
- Exposure: 500 microseconds

**Assay workflow**

Probe hybridization, probe ligation, slide preparation, probe release, extension, and library construction followed the Visium HD Spatial Gene Expression Reagent Kits User Guide 2.0 ([CG001679](/support/spatial-gene-expression-hd/documentation/steps/library-construction/visium-hd-spatial-gene-expression-reagent-kits-user-guide-2-0)).

- Slide serial number: H2-KJHFXHC
- Area: B
- Instrument: Visium CytAssist
- Probe set: Visium Human Transcriptome Probe Set v2.0

**Sequencing**

- Indexing: Dual index plate TS set A; sample index B12
- Sequencing instrument: Illumina NovaSeq X
- Sequencing configuration: 43 bp read 1, 50 bp read 2, 10 bp i7 sample index, and 10 bp i5 sample index
- Sequencing depth: 4.23 B reads

**Analysis**

Space Ranger v4.1.0 was used to map FASTQ files to the reference, detect tissue, align the data to the microscope and CytAssist images, and output feature-barcode matrices for further analysis.

The first iteration of the Pan-Human Azimuth model was trained on scRNA-seq and snRNA-seq data from 23 different human tissues and 380 different cell types. Cancer datasets were excluded from the training process. Performance may be impacted for cancer tissues and other tissue types not used to train the model. For more information, see the [Pan-Human Azimuth page](https://satijalab.org/pan_human_azimuth) from the Satija lab.

**How to view data**

To get started, [download Loupe Browser v9.1](/support/software/loupe-browser/downloads) to explore the Loupe file, or read more about the other [Space Ranger outputs](/support/software/space-ranger/latest/analysis/outputs/output-overview).


CytAssist image

Microscope image

Loupe alignment file

FASTQs

Probe Set CSV

Slide layout file

Barcode mappings parquet

Binned outputs (all bin levels)

Loupe file (with 8 µm bins)

Loupe file (with cell segmentation)

Feature slice H5

Metrics summary CSV

Molecule info H5

Segmented outputs

Spatial outputs

Web summary HTML

Web Summary

Files

Visium CytAssist

Visium

Gene Expression

Protein co-detection

Compact instrument designed to simplify the Visium workflow by facilitating the transfer of analytes from standard glass slides to Visium slides.

HD Spatial Gene Expression

Whole transcriptome probe-based gene expression assay at single cell scale, compatible with FFPE, Fresh Frozen, and Fixed Frozen tissue sections

Space Ranger

Visium Spatial Gene Expression is a next-generation molecular profiling solution for classifying tissue based on total mRNA. Map the whole transcriptome with morphological context in FFPE or fresh frozen tissues to discover novel insights into normal development, disease pathology, and clinical translational research.

Immunofluorescence protein co-detection

Visium CytAssist Gene and Protein Expression

Map the whole transcriptome within the <em>tissue context</em>

Spatial Gene Expression

Spatial gene expression profiling and imaging data of fresh frozen or FFPE tissues.

Analyze your Visium Spatial data with microscope images.

Visium HD Spatial Gene Expression Library, Human Colon Cancer (FF), 11 mm Capture Area

<SupportCallout level={"info"}>This assay requires CytAssist instrument Firmware v2.0.0 or higher. Refer to the [Visium CytAssist with Readiness Test User Guide (CG000542)](/support/instruments/visium-cytassist/visium-cyt-assist-with-readiness-test) for details.</SupportCallout>
<br />

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This document outlines the protocol for generating Visium HD Spatial Gene Expression libraries from FFPE, Fresh Frozen, and Fixed Frozen tissue sections. Key companion documents for use with this document:

1. Visium HD Spatial Gene Expression Protocol Planner (CG000698)
2. Tissue Slide Alignment Instruction, Quick Reference Cards (CG000548)
3. Visium Cassette S3 for Assembly and Disassembly Quick Reference Cards (CG000730)

For use with:

- Visium HD, Human Transcriptome, 6.5 mm, PN-1000675 (4 rxns) / PN-1000673 (16 rxns)
- Visium HD, Mouse Transcriptome, 6.5 mm, PN -1000676 (4 rxns) / PN-1000674 (16 rxns)
- Visium CytAssist Reagent Accessory Kit, PN-1000499
- Dual Index Kit TS Set A, 96 rxns, PN-1000251


This document outlines the protocol for generating Visium HD Spatial Gene Expression libraries from FFPE, Fresh Frozen, and Fixed Frozen tissue sections.