Roy J. Carver
Biotechnology Center

10x Visium spatial transcriptomics

Our facility offers a full range of services for library construction and sequencing with the NovaSeq X Plus, MiSeq, Oxford Nanopore GridION and the 10x Genomics Chromium. The information below is for 10x Visium Spatial Transcriptomics projects only.  Please see our 10x Single Cell page for single cell applications, or return to our main Sample Submission page for all other sample types.

10x Visium spatial transcriptomics projects are processed in the Carver Biotechnology Center (CBC) Cytometry and Microscopy to Omics (CMtO) Core, with final library construction and sequencing performed in the DNA Services lab.

Contact Information:

• CMtO: Project setup and quoting, tissue permeability testing, sectioning and Visium protocol:  

  • Mayandi Sivaguru, Ph.D - Director (sivaguru@illinois.edu) and Kate Janssen (kjans01s@illinois.edu)

• DNA services facility: library preparation and sequencing:

  • Alvaro Hernandez (aghernan@illinois.edu) and Chris Wright (clwright@illinois.edu)

• HPCBio: Bioinformatics processing / Space Ranger / downstream analysis:

  • Chris Fields (hpcbio@biotech.illinois.edu)

Basic Information:

Below, you'll find a host of details and links that you can refer to throughout this process and/or use in your grant submission. You  can explore more about the Visium technology here and find support documentation, software, and data sets for Visium Fresh Frozen (FF) gene expression here and for FFPE Visium here.

If you're writing a grant, please send an email to CMtO and DNA Services, including the funding agency and title of the grant, to get quotes and letters of support.  Include HPCBio if analysis services are also needed.

10x Visium is compatible with fresh frozen OCT embedded tissues and 10x FFPE Visium is compatible with FFPE tissue.  **This is a critical point, as tissue frozen with other methods may result in tissue cracking, crystal formation, or other morphology-altering conditions.  Please see the 10x Tissue Preparation Guide for specific details on how to properly freeze and store your tissue.

10x Visium Technology:

• Visium Technology has expanded to allow for both Fresh Frozen or FFPE processing, with either 6.5mm or 11mm square capture areas depending upon the kit chosen.  We offer ALL 10x Genomics protocols, including the original Fresh Frozen Visium, as well as FFPE Visium, Visium setup with CytAssist, and the latest Visium system, Visium HD!  See more here!

• Each Visium Spatial Gene Expression Slide (Fresh Frozen) includes 4 capture areas (6.5 x 6.5 mm) each defined by a fiducial frame. The capture area has ~5,000 gene expression spots with each spot is ~55 microns  in size. Spots contain oligos that include:

  • Illumina TruSeq Read 1 (partial read 1 sequencing primer)

  • 16 nt Spatial Barcode (all primers in a specific spot share the same Spatial Barcode)

  • 12 nt unique molecular identifier (UMI)

  • 30 nt poly(dT) sequence (captures poly-adenylated mRNA for cDNA synthesis) or probe capture sequence for FFPE samples.

• Distance from center to center of each spot is ~100 microns.

Tissue sections on the capture areas of the Visium Spatial Gene Expression Slide are fixed using methanol. Hematoxylin is used to stain the nuclei, followed by eosin staining for the extracellular matrix and cytoplasm. The stained tissue sections are imaged. 

The same tissue section is permeabilized to release mRNA onto capture spots that contain spatially barcoded oligos fixed to the slide.  mRNAs are converted to cDNAs and then collected for dual-indexed Illumina library construction and sequencing.  The H&E stained image and the spatially barcoded cDNAs are overlaid to allow visualization of the gene expression within the original tissue placement.

Ideally, tissue sections should be no larger than the capture area (6.5mm or 11mm) to avoid covering the fiducial frame that is used to align the RNASeq data with the stained tissue images.  In addition, tissue placed outside the capture area will also simply not generate any additional gene expression data, or could possibly convolute the gene expression data generated.

See this great how-to video on tissue preparation here!

Scheduling:

Contact CMtO to coordinate the experiment.  A planning meeting will be scheduled. The RNA quality of your tissues will be checked first, then the tissue permeability test will be scheduled (fresh frozen only, not FFPE), followed later by the full experiment with tissue sectioning, staining and imaging, mRNA capture, cDNA synthesis, library construction, and sequencing. CMtO can work with you to do test staining of your tissues as needed prior to the initial tissue permeability test.  Be sure to contact them well in advance (2-4 weeks) to get onto their calendar for this work.

Sequencing:

• 10X recommends 25k-50k reads per tissue covered spot, depending upon the system used. 

• For example, if using Visium FF, and the tissue has ~80% coverage per spot, 4k spots covered x 50k reads = 200M cDNA reads per section are needed.  

• Minimum sequencing requires 28nt for read1 reading the spot barcode and UMI, 2 10nt index reads, and 90nt-150nt cDNA reads in read 2 for FF, 50nt for FFPE.  Reads shorter than recommended length are not offered as they can lead to decreased application performance.

Costs:

• Any tissue preparation costs.

• CMtO Service Costs: See CMtO pricing page here.

• DNA Services library preparation ($129/library).

• Sequencing costs:

  • Smaller projects (1 slide) can typically fit on 1 NovaSeq X Plus 10B lanes: $2,190

  • Larger projects should budget 1 or more NovaSeq X Plus 25B lanes: $3,290

Grant Description for FF/FFPE Visium samples: Please contact CMtO for the latest grant description text.

Please contact Dr. Alvaro Hernandez, Director of DNA Services (aghernan@illinois.edu) at 217-244-3480 or Chris Wright, Associate Director of DNA Services (clwright@illinois.edu) at 217-333-4372 to discuss ways the staff can be of assistance in achieving your project goals or to receive a quote for your project, submission forms, grant support or other information needed.

All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.