GSM9301836: lung, WT, naive, rep1; Mus musculus; RNA-Seq
Following sacrifice, the lungs were perfused with cold saline using a 25-gauge needle and a 10 mL syringe via the right ventricle to clear the lung of circulating blood. The lungs were inflated through the trachea with 1mL of digestion mix, consisting of Collagenase Type I (catalog #17100–017, 450 U/mL; Gibco), Dispase (catalog #354235, 5 U/mL; BD Biosciences) and DNase I (catalog #10104159001, 1 mg/ml; Roche) in DMEM/F12. Lungs were dissected out of the thoracic cavity and extraneous tissues removed. Lungs were minced manually with scissors in 1.5 mL Eppendorf tube and then incubated in 4 mL of digestion mix for 25 min at 37 °C with constant agitation. The homogenate was pipetted using 1,000 µl pipette using a wide orifice pipette. This was then centrifuged at 400 g (1,500 rpm) for 7 min. The supernatant was removed, and the pellet resuspended in 5 ml Accutase + 5 mM EDTA for 10 min at 37 °C with constant agitation. This was then filtered using a 70 µm filter, followed by centrifugation of the flowthrough at 400 g for 5-8 min at 4 oC. The supernatant was removed and 2 mL of red blood cell lysis buffer was added, for 2 min at RT. 5 ml of DMEM/F12 +10% FBS was added to stop RBC lysis. Further centrifugation at 400 g for 5-8 minute at 4oC. The pellet was resuspended in 1 ml of FACS buffer (PBS, 1.5% BSA, 2mM EDTA) and filtered through a 40 µm filter (H13680-0040 from Bel-Art). Cells were counted and viability checked using a hemocytometer by Trypan blue exclusion. The concentration was adjusted to 1 x107 cells in 100 ml of FACS buffer for flow cytometry staining and sorting. Library construction was performed as per the manufacturer's instructions.