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Visium HD Spatial Gene Expression Library, Human Breast Cancer, IF (FFPE)

HD Spatial Gene Expression dataset analyzed using Space Ranger 3.1.2

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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.

This dataset is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. 10x citation guidelines available here.