GSM6754356: BHT injury repeat-2; Mus musculus; RNA-Seq
lungs were inflated with an enzyme dissociation solution (450U/mL Collagenase I (Worthington, LS004197), 5U/mL Dispase (Corning, 354235), and 0.33U/mL DNase I (Roche, 10104159001), in DMEM). Separated lung lobes were minced and incubated in enzyme solution at 37°C for 25-35 minutes. Dissociation was quenched with equivalent volume 10% FBS/DMEM and strained through a 100 µm strainer. Cell pellet was resuspended in red blood cell lysis buffer (100 µm EDTA, 10 mM KHCO3, 155 mM NH4Cl) for 2 minutes, followed by quenching with 10% FBS/DMEM and filtration through a 40 µm strainer. Cell pellet was resuspended in 500µL 2%FBS/DMEM. For MACS, cells were incubated for 30 minutes with the following antibodies: CD45 (MiltenyiBiotech, 130-052-301) and CD31 (MiltenyiBiotech, 130-097-418). MACS depletion was performed as per manufacturer protocol using LD columns (MiltenyiBiotech, 130-042-901). Lung cell suspensions were enriched for epithelial and mesenchymal cell populations and resuspended in PBS supplemented with 0.01% BSA. We used 10X genomics, 3'-GEX scRNA-seq (V3.1) protocol to capture cells and prepare libraries. 3'-GEX v3.1 (10X genomics)