Visium CytAssist Spatial Transcriptomics

Getting Started

Project Consultation: We strongly recommend a consultation meeting before starting your first Visium project. Consultations are free for investigators utilizing the ATGC's spatial RNA profiling service. To request a meeting, please contact David Pollock (dpollock@mdanderson.org) or Erika Thompson (ejthomps@mdanderson.org).

Sample Submission

To schedule a submission, please contact David Pollock (dpollock@mdanderson.org).

To submit samples, please complete a Visium CytAssist Spatial RNA profiling Service Request Form and email the completed form (with an active account number and the appropriate signature) to David Pollock. In addition, external submissions require a completed external billing form and an e-copy of the purchase order prior to submitting samples.

Application highlights

• Probe-based assay for tissue mRNA profiling for human or mouse samples
• Capable of running FFPE, fresh frozen, or fixed frozen tissue samples

Technology Overview

Visium Spatial Gene Expression for formalin fixed & paraffin embedded (FFPE) is a probe-based assay allowing simultaneous analysis of molecular and imaging data from FFPE and Frozen tissue sections.

The Visium CytAssist Spatial Gene assay is designed to analyze mRNA in tissue sections sourced from FFPE. It utilizes probes targeting the whole transcriptome. Visium Human Transcriptome Probe Set v2.0 contains approximately 18,000 genes. Visium Mouse Transcriptome Probe Set V1.0 contains approximately 20,000 genes. Each Visium CytAssist Spatial Gene Expression Slide contains Capture Areas with barcoded spots that include oligonucleotides required to capture gene expression probes.

The transcriptome probes are hybridized to the tissue where each left-specific and right-specific probe pair is ligated into one. Tissue slides and Visium CytAssist Spatial Gene Expression v2 Slides are loaded into the Visium CytAssist instrument, where the gene expression-ligated probes are released from the tissue onto the CytAssist slides enabling capture by the spatially barcoded oligonucleotides present on the Visium slide surface. Gene expression libraries are generated from each tissue section and sequenced. Spatial Barcodes are used to associate the reads back to the tissue section images for spatial mapping of gene expression.

Visium Service Workflow for FFPE tissues-customer and ATGC responsibilities

An overview of the workflow is indicated below:

The customer performs or obtains services (through a core facility) for tissue processing, which includes the tissue fixation, embedding, sectioning (slides and curls/scrolls), deparaffinization, staining, imaging, and coverslip removal. For a protocol for these steps, please reference 10X Genomics website. Please review information from their “Visium CytAssist Spatial Gene Expression for FFPE” section for tissue preparation, staining and imaging specifications.

Research Histology Core Lab (RHCL) can perform the tissue sectioning on standard slides (see validated slide types for Visium below). The RHCL offers sectioning, H&E/Immunostaining, imaging, and coverslip removal for the samples. Please see their webpage for contact and sample and submission requirements.

The ATGC will provide the RNA QC, probe hybridization and ligation, permeabilization and barcoding, library creation and sequencing, which includes initial analysis with Space Ranger Pipeline.

RNA will be tested on an Agilent Bioanalyzer RNA chip for its Fragmentation Distribution Value of ≥200 nt (DV200) measurement. DV200 measurements of at least 30% or higher are considered passing to move forward to processing. Samples with DV200 less than 30% may be used (not recommended), but customer assumes the risks (cost, sample, data, etc…). Permission to run suboptimal samples must be given in writing prior to processing.

Sample QC Requirements

Tissue sections should be sized and sectioned in order to fit the probe capture areas of the Visium CytAssist Spatial slides. The Visium CytAssist slides come in two formats, 6.5 mm by 6.5 mm or 11 mm by 11 mm areas. Each Visium CytAssist slide is able of running two tissue samples at once. 

5 um thick FFPE serial slide sections. If the tissue is large and hasn’t been reduced for the 6.5 mm or 11 mm areas on the Visium CytAssist Slide, the approximate area for testing should be annotated on the back of the stained slides (not the tissue-associated side). The annotated area should be clearly visible but easy to remove (with alcohol wipe) before processing. This designated area will be used for all downstream Visium processing steps. Also, slides should not be cover slipped.

One to two (H&E) stained slides and additional unstained serial slide sections (4 or more slides might be required depending on the size of the tissue section) which will be used for RNA extraction and QC. The stained slides (without coverslip) are stored at 4 degrees C (under desiccant) and are stable for two weeks. The unstained slides can be left at room temperature. If applicable, tissue section scrolls/curls or extracted RNA in 1.5 mL microfuge tubes may also be submitted in place of additional serial sections. The QC tubes (scrolls/extracted RNA) should be stored at -80C prior to service submission. 

Slide and Tissue Requirements

Please see the list below of microscope slides that have been validated with this application:

Fisherbrand Premium Plain Glass Microscope Slides- Fisher Scientific cat# 12-544-4

Epredia Shandon ColorFrost Plus Slides- Fisher Scientific cat# 6776214

Poly-Prep Slides- Millipore Sigma cat# P0425

Superfrost Slides- VWR cat# 47311-703

Tissue source should be from either human or mouse. Please no PDX samples.

Tissue section thickness should be as follows:

FFPE: 3-10 µm (5 um is common)

Fresh Frozen: 10-20 µm (10 um is common)

Fresh Fixed Frozen: 10-20 µm (10 um is common)

Tissue thickness will be dependent on the type of tissue.

Tissue sections should be placed on their slides where they are relatively centered in the working area of the slide-15 mm from the short-length slide edge (or frosted area if applicable) and 5 mm from the longer length slide edge. An example is indicated below. Sections outside of this area cannot be run on the CytAssist.

Imaging Requirements

Please follow 10X Genomics guidelines for imaging (website in the customer and ATGC responsibility section above). Slide image IDs should be the same as the actual slide IDs which should also be reflected on the request form. 10X to 40X magnification images can be submitted for this application but usually 10X-20X images are sufficient. It will depend on the tissue being imaged and resolution of the microscope and tiling settings for image stitching. High resolution images (one per slide), which should be submitted as .tif/.tiff files and be shared with the ATGC (by OneDrive; no USBs permitted). We can proceed without the images, but the images created on the CytAssist aren’t quality images and only used for alignment purposes.

Sequencing

Supported sequencers that are available are MiSeq, NextSeq500, and NovaSeq6000. Run format for Visium CytAssist Libraries is 28X50 with dual indexes (10 nt). Depth of sequencing will be approximately 25,000 reads/spot (~5000 spots for 6.5 mm or ~14000 spots for 11 mm Visium CytAssist configurations) multiplied by the total number of tissue-associated spot features on the slide.

Data Output

Raw data fastq files will be used for data analysis in addition to the tissue images submitted with the samples. Our bioinformatics will perform the initial analysis using the Space Ranger Pipeline where the spatial gene expression information will be aligned with the tissue images supplied with the sample as well as the CytAssist images to produce the files which can subsequently be visualized using the Loupe Browser software, which is freeware downloaded from 10X Genomics website.

Contact information

Erika Thompson
Email: ejthomps@mdanderson.org