10x Genomics obtained FFPE Human Glioblastoma tissue block from Avaden Biosciences. The tissue was sectioned as described in Visium CytAssist Spatial Gene and Protein Expression for FFPE – Tissue Preparation Guide (CG000660). 5 µm tissue sections were placed on Superfrost glass slides, deparaffinized, decrosslinked, IF stained, and coverslipped. Sections were imaged and decoverslipped per the Demonstrated Protocol (CG000659).
Sample Description:
- Sex: Female
- Age: 60s
- Date Excised: 2019
- DV200: 47.4
Antibodies used for immunofluorescence (IF) staining:
- Channel 1 = 1:200 DAPI (Thermo Fisher, Cat. No. 62248)
- Channel 2 = 1:100 PCNA, 594 nm, (Biolegend Cat. No. 307914 )
- Blocking buffer: 1X Blocking Buffer (10x Genomics Cat. No. 2000554); 2 U/uL RNase Inhibitor (10x Genomics Cat. No. 2000556)
The IF image was acquired using Olympus Slide Scanning Microscope with these settings:
- Olympus Objective Magnification: 20X UPLXAPO Objective
- Numerical Aperture: 0.8
- ScopeLED light source: Xcite Novum
- Camera: Hamamatsu Orca FusionBT sCMOS
Libraries were prepared following the Visium CytAssist Spatial Gene and Protein Expression Reagent Kits User Guide (CG000494):
- Sequencing instrument: Illumina NovaSeq, flow cell H3TVGDSX7 (lane 4)
- Sequencing depth (Gene): 35,000 reads per spot
- Sequencing depth (Protein): 20,000 reads per spot
- Sequencing configuration: 28bp read 1 (16bp Visium spatial barcode, 12bp UMI), 50bp read2 (probe insert/antibody barcode), 10bp i7 sample barcode and 10bp i5 sample barcode
- Dual-Index set: SI-TS-H11, SI-NT-H11
- Slide: V53J23-361
- Area: B1
Key metrics were:
- Spots detected under tissue: 5,756
- Median genes per spot: 7,629
- Mean reads per spot: 68,906
- Mean Antibody reads per spot: 75,377