Roy J. Carver
Biotechnology Center

10x Genomics Visium Slide Preparation

The 10x Genomics Visium Spatial Gene Expression (GE) system provides total mRNA analysis for intact tissue sections with morphological context. Each GE slide is comprised of four tissue capture areas. The individual areas are 6.5mm x 6.5mm and contain ~5000 barcoded spots measuring 55 µm in diameter and spaced so that the distance between the centers of each spot is 100 µm. Depending on the tissue, an average of 1-10 cells will cover a spot. The spatial barcode assigned to the spot is incorporated during cDNA synthesis and enables gene expression data to be mapped back to its location within the tissue. Data is processed with the 10x SpaceRanger analysis software and can be visualized with the Loupe Browser software.

Research Lab Responsibilities

1. RNA quality must be assessed for each sample prior to use for the Visium experiment. An RNA Integrity Number (RIN) (fresh frozen tissue) >7, or DV200 >50% (FFPE tissue) is recommended by 10X Genomics to proceed. Samples with RNA quality scores below these values may indicate RNA degradation and produce inferior results. CMtO can provide the sample sections to the Primary Investigator (PI) for RNA purification, and DNA Services  can assess the quality.
2. Investigator determines their preferred workflow
   
   1. Fresh Frozen Tissue Options:
      
      1. CMtO performs the full Visium service (thru cDNA and transfer to DNA Services):
         
         1. Frozen tissue embedding in OCT, and cryosectioning for the following:
            
            • RIN preparation by the Investigator
            • Sections placed onto plain glass slides for staining and imaging to verify (by the PI) the presence of the Region of Interest (ROI)
            • Sectioning of an individual sample and positioning onto each Tissue Optimization (TO) slide
            • Sections precisely added to the capture areas of each GE slide
         2. H&E or Immunofluorescent (IF) slide staining (plain glass, TO, GE)
         3. Brightfield and/or IF imaging of slides (plain glass, TO, GE)
         4. cDNA preparation and transfer to DNA Services for library sequencing
      2. Investigator performs any or all of the following preparation steps in their home lab prior to transferring the TO and GE slides (or sample block) to CMtO for processing:
         
         1. Embedding the frozen tissue in OCT blocks.
         2. Any or all sectioning of the tissue block (plain glass slides, TO slide, GE slide).
         3. Staining of the plain glass slides to determine ROI location and morphology acceptability.

1. 2. FFPE tissue options:
      
      1. CMtO performs the full Visium service (thru transfer to DNA Services):
         
         1. Paraffin embedded tissue blocks are sectioned for:
            
            • RNA quality check by the Investigator
            • Staining on plain glass slides for staining to determine ROI location and morphology acceptability
            • Precise placement of sections onto the capture areas of GE slides
         2. H&E or IF staining of plain glass and GE slides
         3. Imagine of plain glass and GE slides
         4. Probe hybridization and release for transfer to DNA services

1. 1. 2. Investigator performs any or all of the following preparation steps in their home lab prior to transferring the GE slide (or sample block) to CMtO for processing:
         
         1. Sectioning of tissue block onto plain glass and/or GE slides
         2. H&E or IF staining of plain slides to visualize section location