Fresh Frozen Visium on CytAssist: Human Breast Cancer, Probe-Based Whole Transcriptome Profiling

10x Genomics obtained fresh frozen (FF) Human Breast Cancer 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: 4.4

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 6000, flow cell HLYH7DSX5 (lane 1-4)
• Sequencing depth: 75,314,377 reads / 15.1% 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-C9
• Slide: V42U20-030
• Area: A1

Key cell metrics were:

• Spots detected under tissue: 1,657
• Median UMI counts per spot: 27,128
• Median genes per spot: 6,782