Spatial Transcriptomics of Murine Lung Tissue

10 μm sections were generated from frozen inflated left lungs using a cryostat and placed on barcoded slides (10x Genomics). The Visium Spatial Gene Expression Slide & Reagent Kit (10x Genomics) was used to generate barcoded cDNA libraries. H&E stained tissues on Visium slides were imaged using a Nikon Eclipse Ti2 inverted microscope. Lung tissue on Visium slides was permeabilized for 14 min to extract mRNA. Barcoded cDNA libraries were sequenced on an Illumina NextSeq 500 instrument using a NextSeq 500/550 High Output Kit v2 (150 cycles) (20024907, Illumina) with the following cycle counts: 28 (read 1), 10 (index 1), 10 (index 2), 90 (read 2). Loupe Browser (v5.0, 10x Genomics) was used to identify which spatial sequencing capture area spots were in contact with tissue. Demultiplexing and alignment was performed with Space Ranger (v2.1, 10x Genomics) and the mm10 2020-A reference transcriptome (10x Genomics). Analysis was mainly performed in R using Seurat (v4.0). Visium datasets were integrated using the SCTransform pipeline, and PCA and UMAP were performed using the top 30 principal components. SPOTlight^{33 (link)} was used to demultiplex Visium data with our integrated scRNA-seq data used as a reference.