title: GSM7505353: Mouse replicate 3; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized with CO2 and lungs were suspended in a digestion buffer containing DMEM, FBS, Collagenase D, DNase, Heparin, CaCl 2 , and MgCl 2 . Lungs were incubated at 37˚C for 30 minutes and dissociated with a gentleMACS dissociator (Miltenyi Biotec), according to the manufacturer's protocol. To isolate cells, lung suspensions were filtered through a 70 μm cell strainer. Sorting for the tetramer-enriched scRNA-Seq was performed using a CD4 + T cell isolation kit (Miltenyi Biotec) followed by tetramer staining and enrichment of tetramer-specific cells with anti-PE microbeads (Miltenyi Biotec). Cells sorted into DMEM with 40% FBS were used for scRNA-Seq sample preparation. After sorting, cell number was confirmed with the Luna II (Logos Biosystems). The appropriate number of cells necessary to achieve a target cell input of 2-10,000 cells was resuspended and used to generate GEMs (Gel Bead-In Emulsion). The Chromium Next GEM Single-cell 5' Gel beads v2 kit (10X Genomics) was used and manufacturer instructions were followed for GEM generation through cDNA synthesis. A Chromium Control was used for GEM generation and barcoding then a thermocycler was used for cDNA synthesis. cDNA was submitted to the University of Minnesota Genomics Center for sequencing on an Illumina Novaseq. Tetramer-enriched scRNA-seq data pooled from three biological replicates.