CellRanger analyzed Visium data

Mouse lung tissues were perfused with PBS and fixed with 10% formalin via tracheal instillation at a constant pressure (15 cm H₂O) and embedded in paraffin wax. Lung tissues were sectioned into 5 μm sections. Tissue sections were placed within the fiducial frame or the etched frames of the Capture Area on the 10X Genomics Visium Spatial slides. Slides were then deparaffinated, decrosslinked and stained with H & E staining kit (Millipore Sigma). Images were acquired under Keyence BZ-X800E slide scanner. The mouse whole transcriptome probe panel is added to the deparaffinized, stained, and decrosslinked tissues. After hybridization, single stranded ligation products were released and then captured on the Visium slides. Probes are extended by the addition of UMI, Spatial Barcode and partial Read 1, followed by library preparation and sample indexed. The library was sequenced on a Hiseq 4000 with pair-end 150bp (Novogene). The raw sequencing data was analyzed by CellRanger 7.0 (10X Genomics) and Seurat V4. Visium data was integrated with scRNA-seq data. The cell annotation was transferred from scRNA-seq.