TRIP Lab, in collaboration with the UWBC Gene Expression Center (GEC) is excited to offer Visium Spatial Gene Expression (10X Genomics), a technology that allows researchers to simultaneously visualize cells and their location in a tissue sample and measure and map gene activity throughout the tissue.
By utilizing IHC/IF and hematoxylin and Eosin (H&E) staining combined with next-generation sequencing (NGS), we are able to examine protein expression, cell morphology, gene expression panels, whole transcriptome analysis, map gene activity, examine mRNA, and spatial relationships between cells.
For Visium Spatial Gene Expression questions and to schedule a meeting to discuss your project and timeline, contact the TRIP Lab at firstname.lastname@example.org or 608-265-9168.
For pricing please see our TRIP Fee Schedule.
To request Visium Spatial Gene Expression services, go to our iLab webpage.
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What does the workflow of a Spatial Gene Expression project look like?
What size should my tissue be?
Tissue should be trimmed to ≤6.5 x 6.5 mm in order to fit onto an 8 x 8 mm Capture Area on the slide. Tissue cannot exceed the walls of the Capture Area.
How should I collect and store my tissue?
The appropriate size tissue should be collected and placed in liquid nitrogen-cooled isopentane prior to OCT embedding or simultaneous OCT embedding and freezing should be performed immediately after tissue is available. Once tissue is in the desired orientation place mold on dry ice until frozen. Place frozen mold in a sealed bag and transfer to -80°C for long-term storage until ready for processing.
The recommended section thickness for most tissue types is 10-12 μm. Some tissues require thicker sections; see list of optimized tissues.
For more information, see the 10x Genomics Tissue Preparation Guide.
Does my tissue need to be optimized?
Yes, 10X Genomics recommends optimizing the permeabilization incubation time for each tissue type being used. Just a few minute variation in incubation can make a difference in your downstream success. As seen in this image, a 12 min incubation results in stronger fluorescent signal compared to the others.
How does Spatial Gene Expression work?
Four 10µ tissue samples are placed on a Visium Gene Expression slide containing 4 Capture Area containing an array of 5,000 gene expression spots, each spot containing barcoded oligo(dT) capture oligonucleotides that imprint the spatial position of a captured mRNA molecule into the downstream cDNA sequencing read.
Staining & Imaging:
Slide is fixed with methanol, stained with Hematoxylin and Eosin, and imaged at 20X using our Aperio whole slide scanner. This image will be used downstream to map the gene expression patterns back to the tissue sections.
*Longley, J., Prasad, A., 2020. University of Wisconsin – Madison, Department of Dermatology
Permeabilization & Reverse Transcription:
A Permeabilization Enzyme is applied to the tissue and incubated for the amount of time determined in optimization. The poly-adenylated mRNA released from the overlying cells is captured by the primers on the spots. Reverse Transcription reagents are added to the tissue and incubated producing spatially barcoded, full-length cDNA from poly-adenylated mRNA on the slide.
Second Strand Synthesis & cDNA Amplification:
Reagents are applied to the tissue to initiate second strand synthesis followed by denaturing and transferring the cDNA from each capture area to an individual tube. Spatially barcoded, full-length cDNA is PCR amplified for library construction.
Library Preparation and Sequencing:
Libraries are prepared in the UWBC Gene Expression Center (GEC), located at 425 Henry Mall, and sequenced on the NovaSeq by UWBC DNA Sequencing Facility, followed by optional analysis by the UWBC Bioinformatics Resource Center.
The resulting data can be analyzed and explored using the freely downloadable Loupe Browser software.
These are the following recommendations for sequencing:
-10x Genomics recommended depth is an under-estimate.
-Evidence suggests 150k-400k reads/spot is required if sensitive UMI detection is desired (depth requirement is highly tissue type-specific).
-Libraries can be sequenced again with the reads aggregated together for increased depth.