Spatial Transcriptomics of Influenza-Infected Lungs

C57BL/6J (Jackson Laboratories) were infected intranasally with 2500 EID₅₀ of IAV PR8 in 30 μl of 1x PBS. Lungs were dissected from the mice 10 days after infection. Sections obtained from four distinct mice were imaged and processed for spatially resolved gene expression using the Visium Spatial Transcriptomics kit (10x Genomics). Lungs were inflated with 300ul of 50% OCT and 50% PBS v/v and immediately snap-frozen in OCT using isopentane that was cooled in a liquid nitrogen bath. For each lung, the same left lobe was harvested and stored at −80°C until cryosectioning. For cryosectioning, samples were equilibrated to −22°C. Exploratory sections were stained with a fluorescent lectin and dapi to determine areas of immune infiltration and remodeling, and then blocks were trimmed to 5×5mm to encompass normal and remodeled areas prior to sectioning directly onto the spatial transcriptomics kit slide. Tissue permeabilization was optimized at 12 minutes on independent lung sections. Libraries were sequenced on the Illumina NovaSeq platform at 28×120 basepairs, and resulting data were processed using SpaceRanger (v1.0.0, 10x Genomics) with manual alignment of fiducial markers, manual tissue identification, and r2-length trimmed to 91bp. Downstream analyses were conducted with Seurat (v3.1.4.9901)^{32 (link)}.