title: Single-cell analysis of mouse lung
design: Single-cell analysis of mouse lung
library_construction_protocol: Lung tissues were removed following transcardial perfusion and washed in cold D-PBS (Biological Industries, 02-023-1A). Trachea was removed and the tissue was immediately injected with an enzyme mix based on previous work [A cellular census of human lungs identifies novel cell states in health and in asthma] (Dispase II 50 caseinolytic u/ml  (Sigma-Aldrich, D4693), Elastase 4.3 u/ml (Worthington Biochem, Worthington, LS002292)), Dnase I 30 μg/ml (Roche), Collagenase A 2 mg/ml (Roche, 10103578001), CaCl2 5 mM) followed by mincing and a brief incubation in a CO2 incubator at 37 ºC. Samples were then transferred to a shaker-incubator for 20 min at 37 ºC, 150 rpm, followed by an immediate addition  of 15 ml of neutralization buffer (DMEM (Biological industries, 01-052-1A), 10% FBS (Rhenium, 10270106).   Samples were then passed through a 40 um strainer twice and pelleted at 400 G, 5 min at 4 °C. Samples were then treated with RBC cell lysis (Sigma, R7757 as according to the manufacturer's protocol) and quenched with (10% FBS, D-PBS). Cells were then pelleted at 400 G, 5 min at 4 °C, resuspended in DMEM and counted. Dead cell removal protocol (Miltney, 130-090-101  ) was applied according to manufacturer protocol in cases when cell viability was lower than 75%, and the flowthrough was pelleted at 400 G ,5 min at 4 °C and resuspended in PBS with 0.05% BSA Cells were counted and loaded on the 10X Genomics Chromium instrument for single-cell sequencing, using 3' V2 chemistry, according to the manufacturer's protocol. cDNA library preparation was were carried out according to the manufacturer's protocol.