10x Genomics obtained fresh frozen (FF) mouse brain tissue blocks from Avaden Biosciences. The tissue was sectioned as described in Visium CytAssist Spatial Gene Expression for Fresh Frozen – Tissue Preparation Guide Demonstrated Protocol (CG000636). Tissue section of 10 µm was placed on a standard glass slide, and H&E-stained following methanol fixation. Sections were coverslipped with 85% glycerol, imaged, decoverslipped, followed by destaining as described in Visium CytAssist Spatial Gene Expression for Fresh Frozen – Fixation, H&E Staining, Imaging & Destaining Demonstrated Protocol (CG000614). The glass slide with tissue section was processed via Visium CytAssist instrument to transfer analytes to a Visium CytAssist Spatial Gene Expression slide with 6.5 mm Capture Area following the Visium CytAssist Spatial Gene Expression Reagent Kits for FFPE User Guide (CG000495). RIN Score: 7.0
The H&E image was acquired using Olympus VS200 Slide Scanning Microscope with these settings:
- Olympus Objective magnification: 20x UPLXAPO Objective
- Numerical Aperture: 0.8
- ScopeLED light source: Xcite Novum
- Camera: VS-264C
- Exposure: 500 µs
Libraries were prepared following the Visium CytAssist Spatial Gene Expression Reagent Kits for FFPE User Guide (CG000495).
- Sequencing instrument: Illumina NovaSeq 6, flow cell HLYH7DSX5 (lane 1-4)
- Sequencing depth: 171,410,389 reads / 38.4% saturation
- Sequencing configuration: 28bp read 1 (16bp Visium spatial barcode, 12bp UMI), 90bp read 2 (transcript), 10bp i7 sample barcode and 10bp i5 sample barcode
- Dual-Index set: SI-TS-G12
- Slide: V42U20-064
- Area: A1
Key cell metrics were:
- Spots detected under tissue: 4,298
- Median UMI counts per spot: 19,627
- Median genes per spot: 6,178