Human Melanoma, IF Stained (FFPE)

10x Genomics obtained FFPE Human Melanoma tissue blocks from Avaden Biosciences. The tissue was sectioned as described in Visium CytAssist Spatial Gene Expression for FFPE – Tissue Preparation Guide Demonstrated Protocol (CG000518). Tissue sections of 5 µm was placed on a standard glass slide, deparaffinized followed by immunofluorescence (IF) staining. Sections were coverslipped with 85% glycerol, imaged, decoverslipped, followed by dehydration & decrosslinking Demonstrated Protocol (CG000519). The glass slide with tissue section was processed via Visium CytAssist instrument to transfer analytes to a Visium CytAssist Spatial Gene Expression slide. The probe extension and library construction steps follow the standard Visium for FFPE workflow outside of the instrument.

• Diagnosis: Skin, Malignant Melanoma

Antibodies used for immunofluorescence (IF) staining:

• Channel 1 = 1:200 DAPI (Thermo Fisher, Cat. No. 62248)
• Channel 2 = 1:100 Vimentin, 488 nm, (Biolegend Cat. No. 677809)
• Channel 3 = 1:100 PCNA, 594 nm, (Biolegend, Cat. No. 307914)
• Channel 4 = 1:100 CD8a, 647 nm, (Biolegend, Cat. No. 372906)
• Blocking buffer: 1X Blocking Buffer (10x Genomics Cat. No. 2000554); 2 U/uL Roche Protector RNase Inhibitor

The IF  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: Hamamatsu ORCA-Fusion
• Exposure: 2.5 ms (DAPI), 300 ms (ATTO488), 150 ms (ATTO590), 500 ms (ATTO647) 

Libraries were prepared following the Visium CytAssist Spatial Gene Expression Reagent Kits for FFPE User Guide (CG000495).

• Sequencing instrument: Illumina NovaSeq, flow cell HNNNMDSX3 (lane 1-4)
• Sequencing depth: 55,952 reads per spot
• 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-C1
• Slide: V42A20-355
• Area: A1

Key metrics were:

• Spots detected under tissue: 3,458
• Median genes per spot: 7,598
• Median UMI counts per spot: 27,894