Spatial Gene Expression Assays (10x Genomics, Visium)
The Visium spatial transcriptomics platform allows investigators to combine histological information with gene expression information from FFPE, fixed-frozen, or fresh-frozen tissue samples to produce spatially resolved gene expression data.Visium Assays
10x Genomics offers several Visium kits. The kits utilize two different methods to obtain spatial transcriptomics data. The following are currently offered through the Single Cell Genomics Facility.- CytAssist method
- Direct-mount methods
CytAssist Method
The CytAssist instrument allows investigators to conveniently submit pre-screened tissue, sectioned onto plain glass slides, to the SCGF avoiding the need to handle expensive, oligo arrayed Visium slides. The tissue on plain glass slides is stained (either H&E or IF) and imaged by the SCGF using the Keyence BZ-X810 (see exceptions below). Once histological information is recorded, the gene expression information is acquired. Either human or mouse whole transcriptome probe panels (consisting of probe pairs for each targeted gene) are subjected to the tissue enabling probe-pair hybridization and ligation. Post ligation, the plain glass slides and Visium slide are mounted into the CytAssist instrument. Here the ligated probe-pairs are transferred from the tissue on the plain glass slides to the barcoded-array capture areas on the Visium slide maintaining spatial configurations. After the probe-pairs are successfully transferred to the Visium slide, the slide is removed from the instrument, and the probes are extended to incorporate the spatial barcoding information. Lastly, the barcoded products are released from the Visium slide, collected in a tube, and further processed into sequencing libraries.CytAssist Assays
There are several different assays offered based on sample preparation and species.
CytAssist FFPE Assays:
Are designed to obtain data from fixed and archived samples of various qualities embedded in paraffin.
- Spatial Gene Expression Human or Mouse Transcriptome 6.5mm, 2 rxns per slide
- Spatial Gene Expression Human or Mouse Transcriptome 11mm, 2 rxns per slide
- Spatial Gene Expression Human or Mouse Transcriptome 6.5mm HD, 2 rxns per slide
Are designed to obtain data from unfixed or fixed tissue embedded in OCT.
- Spatial Gene Expression Human or Mouse Transcriptome 6.5mm, 2 rxns per slide
- Spatial Gene Expression Human or Mouse Transcriptome 11mm, 2 rxns per slide
- Spatial Gene Expression Human or Mouse Transcriptome 6.5mm HD, 2 rxns per slide
CytAssist Sample Requirements
A tissue block quality control (QC) assessment is required for both FFPE, fresh-frozen, and fixed-frozen samples before beginning a Visium experiment. Tissue scrolls can be submitted to the SCGF, and the staff will isolate, purify, and QC check the RNA from tissue. See “CytAssist Checklist” for more information.Tissue sections must be placed on plain glass slides that are provided by SCGF staff. The tissue sections placed onto plain glass slides can be larger than the active area of the Visium slide (11 x 11mm or 6.5 x 6.5mm), but only a selected 11 x 11mm or 6.5 x 6.5mm region of tissue will be transferred to the active area of the Visium slide to capture gene expression data. Investigators are encouraged to cut serial sections from their tissue block, stain the serial slide, identify a region-of-interest, and mark the region-of-interest on the back of the serial slide. The SCGF staff will use the serial slide to identify the region of interest on the unstained, experimental slide.
CytAssist Checklist
- Submit tissue scrolls to SCGF for QC check.
- QC requirements:
a. FFPE Samples: Tissue block QC DV200 score >30%
b. Fixed-Frozen Samples: Tissue block QC DV200 score >30%
c. Fresh-Frozen Samples: Tissue block QC RIN score >4 - Obtain user rights to Visium scheduling calendars in Stratocore.
- Schedule a CytAssist experiment.
Direct-Mount Method
The direct-mount method is strictly for fresh-frozen, OCT-embedded samples. This method requires that the tissue sections be placed directly onto the active areas of the Visium slide. The Visium slide is then fixed, stained, and permeabilized (based off required preliminary data obtained using the Tissue Optimization Slide). After permeabilization, the mRNAs are released and captured by Poly(dT)VN primers found in the spatial barcoding spots. cDNA is synthesized in a reverse transcription reaction that produces full-length, spatially barcoded cDNA. Next, second strand synthesis, denaturation, and cDNA purification are performed. Once the full-length cDNA is purified, it is amplified and constructed into a library.Direct-Mount Assays
- Tissue Optimization Slide 6.5mm Kit, 6 conditions per slide
- Gene Expression Slide 6.5mm Kit, 4 samples per slide
Direct-Mount Sample Requirements
A tissue block quality control (QC) assessment is required before beginning a Visium experiment. Tissue scrolls can be submitted to the SCGF, and the staff will isolate, purify, and QC check the RNA from tissue. See “Direct-Mount Checklist” for more information.The direct-mount method requires a one-time tissue optimization experiment for each tissue-type submitted to the SCGF. The Visium Tissue Optimization Slide is used to perform a time-course experiment to determine the optimal permeabilization time to release sufficient mRNA from the tissue. This time is recorded and is used for all downstream experiments using this tissue-type.
Direct-Mount Checklist
- Submit tissue scrolls to the SCGF for QC check.
- QC requirements:
a. Fresh-Frozen Samples: Tissue block QC RIN score >7 - Obtain user rights to Visium scheduling calendars in Stratocore. Email SCGF staff.
- Schedule a Tissue Optimization Slide experiment and obtain results.
- Schedule a Gene Expression Slide experiment.
It is best to submit a tissue of optimal quality to receive high quality data. If poor-quality tissues are used for experiments, poor-quality data will be generated. The SCGF will advise on using a tissue block based on the data generated during the QC check. Lastly, it is encouraged to QC check multiple tissue blocks to determine which are most suitable for an experiment.
