10x Genomics obtained FFPE human prostate tissue from Indivumed Human Tissue Specimens. Original diagnosis with adenocarcinoma. The tissue was sectioned as described in Visium Spatial Gene Expression for FFPE – Tissue Preparation Guide Demonstrated Protocol (CG000408). Tissue sections of 10 µm were placed on Visium Gene Expression slides, then stained follwoing Deparaffinization, Decrosslinking, Immunofluorescence Staining & Imaging Demonstrated Protocol (CG000410).
- Block 1E333_Tp11 Section 1
- Stage II
- Total Gleason score: 7
- Sex: Male
Antibodies used for immunofluorescence (IF) staining:
- Channel 1 = 1:100 Iba1/AIF-1 (Cell Signalling, Cat. No. 36618S)
- Channel 2 = 1:100 Vimentin (Cell Signalling, Cat. No. 9856S)
- Channel 3 = 1:5000 DAPI (Thermo Scientific, Cat. No. 62248)
- Blocking buffer: 1X PBS; 2% BSA; 0.1% Tween 20; 1 U/uL Roche Protector RNase Inhibitor (based on CoA)
The IF image was acquired using Metafer Slide Scanning Microscope from MetaSystems with these settings:
- Zeiss Plan-Apochromat 20x objective
- Numerical Aperture: 0.8
- Zeiss Colibri 7 LED light source
- MetaSystems CoolCube 4c camera
Libraries were prepared following the Visium Spatial Gene Expression Reagent Kits for FFPE User Guide (CG000407 Rev A).
- Sequencing instrument: Illumina NovaSeq, flow cell H25NNDMXY (lane 1)
- Sequencing depth: 29,191 reads per spot
- Sequencing configuration: 28bp read 1 (16bp Visium spatial barcode, 12bp UMI), 50bp read2 (transcript), 10bp i7 sample barcode and 10bp i5 sample barcode.
- Dual-Index set: SI-TS-D4
- Slide: V11J26-076
- Area: D1
Key metrics were:
- Spots detected under tissue (Loupe Manual Alignment): 3,460
- Median genes per spot: 5,001
- Median UMI counts per spot: 11,444