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Human Melanoma, IF Stained (FFPE)

Spatial Gene Expression dataset analyzed using Space Ranger 2.0.0

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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

This dataset is licensed under the Creative Commons Attribution license.