title: GSM8213190: Bleo-35-days 1; Mus musculus; RNA-Seq
library_construction_protocol: Lung tissues from uninjured sham (n=3), as well as from those harvested at 14 days (n=2) and 35 days (n=2) after bleomycin challenge, were collected and the lung tissues dissociated into a single-cell suspension. Briefly, mice were anesthetized with FATAL-PLUS solution (Vortech pharmaceuticals, Dearborn, MI, USA). After opening the thoracic cavity of mice, the lungs were perfused with 10 mL of sterile cold PBS through the right ventricle of the heart. The exposed trachea was cannulated with a 20G catheter and injected with 2 mL of digestion solution (PBS containing 80 U/mL collagenase type II (Worthington Biochemical, Lakewood, NJ, USA) and 2 U/mL Dispase II (Sigma-Aldrich)), which was followed by clogging with forceps for 2 min. The lung lobes were dissected from the trachea, finely minced, and incubated with 20 mL digestion solution for 1 h in a 37 °C incubator with shaking followed by passaging through an 18G needle 4–5 times and an additional 15 min incubation at 37 °C. After passing through a 21G needle, the cell suspension was filtered through 70 and 40 μm mesh cell strainers and centrifuged at 400× g for 5 min to collect the isolated cells. Red blood cells were lysed using lysis buffer (Sigma-Aldrich). Hematopoietic (CD45+ve) cells were depleted using magnetic cell separation (MACS). Cells were then centrifuged (500×g, 10 min, 4 °C) and resuspended in 0.2 ml of 1X PBS buffer containing 0.04% BSA.  Cell suspensions were submitted to the Single-Cell Sequencing Core (Boston University) for processing. scRNA-seq libraries were generated in BU DOM Single Cell sequencing core facility and sequenced by BU Microarray and Sequencing Resource Core Facility according to 10x Genomics protocol