Biomaterials
A human female pancreas tissue block (FFPE) was obtained from Avaden Biosciences.
Sample preparation
A 5 µm section was taken from the FFPE tissue block with a microtome (Epredia HM355S). Sectioning, deparaffinization, H&E staining and imaging followed the Visium HD FFPE Tissue Preparation Handbook (CG000684).
Imaging
- Image type: H&E
- Microscope: Olympus VS200 Slide Scanner
- Objective magnification: 20X
- Numerical Aperture: 0.8
- ScopeLED light source: VS200 LED, Olympus integrated bright field source
- Camera: iDS VS-264C, Olympus scanner integrated camera
- Exposure: 500 microseconds
Assay workflow
Probe hybridization, probe ligation, slide preparation, probe release, extension, and library construction followed the Visium HD Spatial Gene Expression Reagent Kits User Guide (CG000685).
- Slide serial number: H1-HBNMBMC
- Area: D-1
- Instrument: Visium CytAssist
- Probe set: Visium Human Transcriptome Probe Set v2.0
Sequencing
- Indexing: Dual index plate TS set A; sample index D2
- Sequencing instrument: Illumina NovaSeq 6000
- Sequencing configuration: 43 bp read 1, 50 bp read 2, 10 bp i7 sample index, and 10 bp i5 sample index
- Sequencing depth: 418 M reads
Analysis
Space Ranger v3.0 was used to map FASTQ files to the reference, detect tissue, align the data to the microscope and CytAssist images, and output feature-barcode matrices for further analysis.
How to view data
To get started, download Loupe Browser v8.0 to explore the Loupe file, or read more about the other Space Ranger outputs.