10x Genomics Visium

General Features

A Visium Gene Expression (GEX) slide is comprised of two or four tissue capture areas. Each area is 6.5mm x 6.5mm and contains ~5000 barcoded spots measuring 55 µm in diameter and spaced so that the distance between the centers of each spot is 100 µm.

Image provided by 10x Genomics

Fresh-frozen tissue sections are placed on the slide, which is H&E stained and imaged in bright field. Depending on the tissue, an average of 1-10 cells will cover a spot. The spatial barcode assigned to the spot is incorporated during cDNA synthesis and enables gene expression data to be mapped back to its location within the tissue. Data is processed with the 10X SpaceRanger analysis software and can be visualized with the Loupe Browser software.

Sample Requirements

Fresh/Frozen tissue

• Freshly obtained tissue should be snap frozen to prevent RNA degradation.
• The recommended freezing method uses an isopentane and liquid nitrogen bath
• Frozen tissue should be embedded in Optimal Cutting Temperature (OCT), a freezing and embedding compound (can be done simultaneously with freezing process).
• RNA quality of the tissue block should be assessed prior to sectioning. The RNA Integrity Number (RIN) should be ≥ 7. 
• Each new tissue type for a lab is required to undergo a tissue optimization step so that the correct permeabilization time can be established prior to scheduling a Visium GEX appointment.

FFPE tissue

• Species Specific – only human and mouse samples currently supported.
• 10x Genomics recommended tissue prep guide
• Tissue sections need to be ≤6.5 x 6.5 mm.
• Recommended section thickness is 5 µm.
• RNA quality of the block should be assessed prior to sectioning. The DV200 should be ≥ 50%. 
• Each FFPE block is required to undergo a tissue adhesion test to assess the risk of the FFPE section detaching during the Visium Spatial Gene Expression workflow.

Reagents

AGC Visium Fresh/Frozen Reagents January 2020 – present

AGC Visium FFPE Reagents June 2021 – present

Image provided by 10x Genomics

The core stocks Visium slides. To request a slide pick-up day, please initiate a new Spatial Transcriptomics Service Request in MiCores. This generates a service request number for your Visium project as well as a sample (slide) identification number for tracking purposes, e.g. 1234-MM-S1. Please note that the slide has a defined orientation, with the serial number information located at the top. Depending on the slide, each will contain 2-8 capture areas. Each slide will generate multiple sets of files (one per capture area) and data files for each capture area will be labeled:

• Visium Gateway slides will generate two data sets: 1234 -MM-S1-A and 1234-MM-S1-B.
• Visium Spatial slides will generate four data sets: 1234 -MM-S1-A and 1234-MM-S1-B, 1234-MM-S1-C, and 1234-MM-S1-D.

*** note the first capture area is closest to the serial number at the top of the slide***

Visium Workflow

Visium Gene Expression is essentially a set of slides, reagents, and software tools that enable a person to do whole transcriptome analysis on a tissue section. Thus, there are a variety of ways you can opt to use the platform and work with the Advanced Genomics Core. 

Customer sectioned (most popular) – Investigators section tissue onto the Visium slide. Once tissue is placed on the slide, the slide is stable at -80 for up to four weeks so it can be easily transported on dry ice to the AGC.  AGC staff will perform H&E staining, imaging, library prep, sequencing, and SpaceRanger data processing. 

Work with one of the tissue processing cores on campus – Investigators can coordinate with their preferred tissue facility to section onto the Visium slide. You can also work directly with that or another facility to stain and image the slide, but you will need to meet the 10X imaging requirements. This option needs to be tightly coordinated with the AGC for hand-off of the slides. There is not a safe stopping point so AGC staff need to be prepared to accept imaged slides and proceed with the workflow.

Tissue Submission – Tissue processed following the recommended protocols can be submitted directly to the AGC for sectioning, staining/imaging, library prep, sequencing, and data processing. The cryomold used for embedding should be of appropriate size to fit the tissue sample. Preferred size for cryoblock submission is 10 mm.

Self Service – The Visium platform does not require specialized instrumentation. Reagents can be purchased directly from 10x Genomics and users can prep samples in their own lab space. These samples are submitted to the AGC as user-made libraries ready for Next Generation Sequencing.

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Helpful Documents

• Tissue Prep Guide
• Visium Slide Reset Protocol 
• Imaging Guidelines