10x Genomics obtained FFPE mouse brain tissue from Adlego and embedded by BioIVT. The tissue was sectioned as described in Visium Spatial Gene Expression for FFPE – Tissue Preparation Guide Demonstrated Protocol (CG000408). Tissue sections of 5 µm were placed on Visium Gene Expression slides, then stained following Deparaffinization, Decrosslinking, Immunofluorescence Staining & Imaging Demonstrated Protocol (CG000410).
- Sex: Male
- Age: >8 weeks
- Strain: C57BL/6
- Section Orientation: Coronal
Antibodies used for Immunofluorescence staining:
- Channel 1 = 1:100 NeuN (Abcam, Cat. No. ab190565)
- Channel 2 = 1:200 GFAP (Sigma-Aldrich, Cat. No. C9205-.2ML)
- Channel 3 = 1:5000 DAPI (Thermo Scientific, Cat. No. 62248)
- Blocking buffer: 1X PBS; 2 % BSA; 0.1 % Tween 20; 1 U/uL Roche Protector RNase Inhibitor (based on CoA)
The IF image was acquired using Metafer Slide Scanning Microscope from MetaSystems with these settings:
- Zeiss Plan-Apochromat 20x objective
- Numerical Aperture: 0.8
- Zeiss Colibri 7 LED light source
- MetaSystems CoolCube 4c camera
Libraries were prepared following the Visium Spatial Gene Expression Reagent Kits for FFPE User Guide (CG000407 Rev A).
- Sequencing instrument: Illumina NovaSeq, flow cell HCYTVDSX2 (lane 1-2)
- Sequencing depth: 30,337 reads per spot
- Sequencing configuration: 28bp read 1 (16bp Visium spatial barcode, 12bp UMI), 120bp read2 (transcript), 10bp i7 sample barcode and 10bp i5 sample barcode
- Dual-Index set: SI-TS-A9
- Slide: V11J26-128
- Area: A1
Key metrics were:
- Spots detected under tissue: 2,438
- Median genes per spot: 6,056
- Median UMI Counts per spot: 17,096