GSM8735738: Young (2 month old) C57BL/6 lung fibroblasts and epithelium; Mus musculus; RNA-Seq
After euthanasia and cardiac perfusion with PBS, mouse lungs were tracheally insufflated with a digestion cocktail of Collagenase Type I (225 U/ml, Thermo Fisher), Dispase (15 U/ml, Thermo Fisher) and Dnase (50 U/ml, Sigma) and removed from the chest. The lungs were incubated in digestion cocktail for 1 hour at 37C with continuous shaking. The mixture was then washed with FACS buffer (2% FBS and 1% Penicillin-Streptomycin in DMEM) and passed through a 70 μm cell strainer. Cells were pelleted at 550xg for 5 minutes and then resuspended in red blood cell (RBC) lysis buffer for 2 minutes. After RBC lysis cells were passed through a 40 μm cell strainer. Cell suspensions were blocked with mouse FcR blocking reagent (Miltenyl Biotec 130-092-575) for 15 minutes and then incubated with the appropriate fluorescently-conjugated antibodies in sorting buffer for 30 min at 4C. After washing with FACS buffer cells were sorted. Doublets and dead cells were excluded based on forward and side scatter and DRAQ7, respectively. The following antibodies were used for staining: anti-CD45 PE-Cy7, anti-CD31 PerCP, and anti-Epcam BV421. FACS was performed on a BD FACS Aria using FACSDiva Software. cDNA libraries were prepared for sequencing using standard protocol for the 10x Single Cell instrument.