GSM9366393: Lung, infected, no antibiotics; Mus musculus; RNA-Seq
At the indicated timepoints, lung lobes were harvested, minced, and incubated for 30 minutes at 37°C in dissociation media (RPMI-1640 with 0.7 mg/ml collagenase XI and 30 mg/ml type IV bovine pancreatic DNase). After incubation, lobes were passed through a wide bore pipet tip and filtered through a 40 μm filter. Single cell lung suspension was then counted, aliquoted, and blocked with CD-32 Fc block (BD cat #553142) for 20 minutes on ice. After 2% FBS staining buffer wash, cells were incubated with the conjugated primary antibodies anti-CD45 (BioLegend cat # 103106) and anti-Ter119 (BioLegend cat# 116211). In the same manner, fluorescence minus one controls were blocked and stained with the appropriate antibody controls. Cells from individual mice were then incubated with identifiable hashtags, resuspended in staining buffer, and treated with PI viability dye. 2500 CD45 positive, 5000 CD45 negative, Ter119 negative, viable cells were collected by fluorescence associated cell sorting using a 70 μm nozzle on a 4-laser FACSAria III Cell Sorter. Both single and fluorescence-minus-one controls were used for compensation. ScRNA-seq libraries were generated using the 10X Chromium platform 5' library preparation kits (10X Genomics) following the manufacturer's recommendations and targeting 10,000 - 20,000 cells per sample.