GSM8574722: PBS-treated, scRNA; Mus musculus; RNA-Seq
For lung cells, after euthanasia, the mice were perfused with 10 ml cold PBS through the right ventricle to flush out blood cells in the lung. Then the mice were inflated through the trachea with 2 ml digestion solution (Collagenase IV 0.2 mg/ml, FBS 5% and DNase I 60 U/ml in RPMI-1640 Media. FBS, Gibco, 10099141; RPMI-1640, Invitrogen, 22400089). The lungs were removed and minced into small pieces in 25 ml digestion solution for 30 mins at 37°C with shaking and frequent agitation. After digestion, the cells were filtered through a 70 μm strainer, centrifuged at 500 g for 5 mins at 4°C to discard supernatant. Next, cells were incubated in 1 ml Red Blood Cell lysis buffer (eBioscience, 00-4333-57) at room temperature for 5 mins. 9 ml cold PBS was added and centrifuged at the speed of 500 g for 5 mins at 4°C to discard supernatant. After red blood cell lysing, the cells were washed twice. The isolated cell suspension was processed using the 10x Chromium system, with 13,000 cells loaded during the generation of GEMs. The library preparation followed the guidelines provided in the Single Cell 3' Gene Expression Kit (v3.1) manual.