Biomaterials
A human female breast cancer (ductal carcinoma in situ, sample quality 37.2 DV200) FFPE tissue block 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, staining and imaging followed the Visium HD FFPE Tissue Preparation Handbook (CG000684).
Antibodies used:
- Channel 1 - Alexa Fluor 594 anti-CD45RO (clone UCHL1, Biolegend 304254, dilution 1:100)
- Channel 2 - Alexa 488 anti-CD20 (clone EPR459Y; Abcam ab198941, dilution 1:100)
- Channel 3 - DAPI
Imaging
- Image type: IF
- Microscope: Olympus VS200 Slide Scanner
- Objective magnification: 20X
- Numerical Aperture: 0.8
- ScopeLED light source: Excelitas X-Cite NOVEM-L
- Camera: Hamamatsu Orca FusionBT sCMOS (2304 x 2304 pixels (16bits), 6.5um pixel size, 95% QE, 89.1 fr/sec, ~1Gb/sec)
- Exposure: DAPI (ex 385 nm, em 447 nm): 0.75 microseconds; 488 (ex 466 nm, em 515 nm): 125 microseconds; 590 (ex 586 nm, em 628 nm): 50 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-X632C36
- 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 B8
- 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: 361.5 M reads
Analysis
Space Ranger v3.1.2 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 v8.1 to explore the Loupe file, or read more about the other Space Ranger outputs.