Skip to main content

10x Visium spatial transcriptomics

Our facility offers a full range of services for library construction and sequencing with the NovaSeq 6000, MiSeq, Oxford Nanopore GridION and the 10x Genomics Chromium. The information below is for 10x Visuim Spatial Transcriptomics projects only.  Please return to our main Sample Submission page for all other sample types.

10x Visium spatial transcriptomics projects are processed as a joint collaboration between the Carver Biotechnology Center (CBC) DNA Services facility and the CBC Cytometry and Microscopy to Omics (CMtO) Core.

Contact Information:

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 gene expression here.

If you're writing a grant, please send us an email, including the funding agency and title of the grant, and we can send quotes and letters of support.  

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.

Workflow:

  • The PI is required to purchase the 10X kit (Visium Spatial Gene Expression Slide & Reagents Kit) and schedule with the two Core facilities.  Kits come in 4 reaction (1 slide) or 16 reaction (4 slide) options.

  • CMtO facility personnel perform the tissue permeability testing and first part of the Spatial protocol:  cut and mount the sections, perform the initial staining, imaging, and mRNA capture to cDNA synthesis. 

  • DNA Services receives the cDNA and proceeds with library construction and sequencing.

  • Advanced planning and coordination are required to schedule the experiment, microscopy time, and DNA Services time together.

10x Visium Technology:

  • Each Visium Spatial Gene Expression Slide includes 4 capture areas (6.5 x 6.5 mm), each defined by a fiducial frame (fiducial frame + capture area is 8 x 8 mm). The capture area has ~5,000 gene expression spots, each spot is ~55 microns with primers 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).

  • 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 x 6.5 mm) 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 both CMtO and the DNA Services facility to coordinate the experiment.  A planning meeting will be scheduled. The tissue permeability test will be scheduled first, followed later by the full experiment with tissue sectioning, staining and imaging, mRNA capture, cDNA synthesis, library construction and sequencing.  These should both be scheduled at least 2 weeks in advance to ensure the labs will have staff available to process your samples.  CMtO can work with you to do test staining of your tissues as needed prior to the initial tissue permeability test.

Sequencing:

  • 10X recommends 50k reads per tissue covered spot. 

  • If the tissue has ~80% coverage per spot, 4k spots covered x 50k reads = 200M cDNA reads per section.  

  • Minimum sequencing requires 28nt for read1 reading the spot barcode and UMI, 2 10nt index reads, and 90nt cDNA reads in read 2.  Reads shorter than 90nt are not recommended by 10x Genomics as they can lead to decreased application performance.

Costs:

  • Any tissue preparation costs.

  • CMtO Core rate, $70/hour (tissue optimization); $75/hr (Gene Expression).

    • Tissue Optimization: ~4 hrs / slide

    • Gene Expression Slide (4 samples): ~6-8 hrs / slide

  • Visium Tissue Optimization Slides, $820 per slide. One slide per tissue type (you may need 2 slides if tissue morphology changes, ie tumor/normal).

  • Visium Gene Expression Slide and Reagent kit:

    •  4 samples: $4,805 (this is one slide, single use, with 4 squares, so plan on 4 tissue sections).

    • 16 samples: $17,472 (this is 4 slides, each slide is single use with 4 squares).

  • DNA Services library preparation ($30/library) and sequencing costs (variable, see examples below).

Example 1: 1 slide, 4 sections (4 libraries), 80% coverage = 4k spots @ 50k reads/spot:

  1. 10x Visium 4 reaction kit: $4,805
  2. Tissue optimization slide: $820
  3. CMtO tissue optimization labor, 1 slide @ 4 hrs each* ($70 x 1 x 4): $280
  4. CMtO hourly rate of $75 for 1 slides @ 8hrs per slide* ($75 x 1 x 8): $600
  5. Library construction: $30 x 4: $120
  6. Sequencing on 2 SP 28x150nt lanes: $4,520 -or- Sequencing on 2 SP 28x91nt lanes: $3,460
  • Total: $11,145 (150nt cDNA reads) -or- $10,085 (90nt cDNA reads)

Example 2: 4 slides, 16 sections (16 libraries), 80% coverage = 4k spots @ 75k reads/spot:

  1. 10x Visium 16 reaction kit: $17,472
  2. Tissue optimization slides (2): $1,640
  3. CMtO tissue optimization, 2 slides @ 4 hrs each* ($70 x 2 x 4): $560
  4. CMtO hourly rate of $75 for 4 slides @ 8hrs per slide* ($75 x 8 x 4): $2,400
  5. Library construction: $30 x 16: $480
  6. Sequencing on 2 S4 2x150nt lanes: $11,860
  • Total: $34,412 (150nt cDNA reads)

*hourly rate is estimated for maximum time, final cost may be reduced.

Grant Description for FF Visium samples:

The Carver Biotechnology Center DNA Services laboratory, Cytometry and Microscopy to Omics Core (CMtO), and the High-Performance Computing in Bioinformatics units (Roy J. Carver Biotechnology Center) have received training from 10X Genomics, are fully equipped, and work seamlessly to offer the Visium Spatial transcriptomics service on campus, see attached letters of support. Briefly, frozen tissues are sectioned and placed onto a Tissue Optimization Slide with eight capture areas, each having thousands of spots with poly-dT capture probes. Tissue sections are permeabilized across a time-course from 0 to 36 minutes. The mRNAs from the tissue anneal to the oligos, are converted to cDNA by reverse transcription with a fluorophore and the sections are evaluated with the Zeiss Axiovert 200M microscope. The permeabilization condition producing the brightest image with the least diffusion is chosen for the processing of the tissues. Once optimal conditions have been identified, tissue sections are placed onto the Visium spatial Gene Expression slide, which contains 4 capture area squares 6.5mm x 6.5mm. Each square has approximately 5,000 spots with barcoded poly-dT oligos. Tissue sections are fixed, stained with hematoxylin and eosin (H&E) and visualized with the custom LSM-980 microscope. After permeabilization, messenger RNAs are converted into spatially-barcoded cDNAs. The double-stranded-barcoded cDNAs are then denatured, pooled and converted into a sequencing-ready and dual-indexed library. The libraries are sequenced in a NovaSeq 6000 to a length of 28nt (read 1, contains the spot barcode and unique molecular identifier used for removing PCR duplicates), 10nt for each index (libraries contain unique dual indexes to prevent index switching) and a minimum of 90nt for read 2 (the cDNA read) to a minimum depth of 50,000 cDNA read 2 per spot. Data is processed and visualized using Space Ranger Analysis Pipelines and Loupe browser.  Visium uses the cDNA barcodes to associate the transcripts to an X-Y coordinate on the slide, which can then be used to overlay the H&E-stained image with the transcript information from a spatial viewpoint.


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.