Biomaterials
Fresh frozen human female breast cancer tissue (ductal carcinoma in situ; RIN: 7.7) was obtained from BioIVT.
Sample preparation
A 10 µm section was taken with a cryostat (Epredia CryoStar NX70). Tissue block preparation, sectioning, H&E staining, and imaging followed the Visium HD Fresh Frozen Tissue Preparation Handbook (CG000763).
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: H1-649ZJJX
- Area: A-1
- Instrument: Visium CytAssist
- Probe set: Visium Human Transcriptome Probe Set v2.0
Sequencing
Visium HD libraries were amplified to append Ultima-compatible sequences and pooled before sequencing. For additional information regarding making 10x libraries Ultima-compatible, please reach out to technical support at Ultima Genomics.
- Indexing: Libraries were adapted for sequencing on the UG 100™ sequencing platform via conversion PCR using Ultima-specific (UG-tR1 and UG-srR2) indexing primers
- Sequencing instrument: Ultima UG 100™
- Sequencing configuration: sequenced via the UG 100 Solaris™ Flex workflow
- Sequencing depth: 2 billion (downsampled)
Analysis
Space Ranger v3.1.2 was used to map reads 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 v8.1 to explore the Loupe file, or read more about the other Space Ranger outputs.