GSM9392321: Lung adventitial fibroblasts from young B6 mouse Y2; Mus musculus; RNA-Seq
After euthanasia with isoflurane the chest cavity was opened and the lungs were perfused with PBS via the right ventricle. The lungs were then 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 lung was incubated in digestion cocktail for 45 minutes 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. Cells were FcR blocked for 15 minutes and then incubated for 30 minutes with the following biotinylated antibodies to deplete lineage+ cells: Ter119-biotin, CD146-biotin, CD45-biotin, CD31-biotin, Epcam-biotin. Lineage+ cells were then removed via magnetic bead using the EasySep Mouse Streptavidine RapidSpheres Isolation Kit (StemCell Technologies #19860). Remaining cells were then incubated with the following fluorescently-conjugated antibodies in sorting buffer for 30 min at 4C: anti-CD45 BV421, anti-CD31-eFluor450, anti-Epcam-BV421, anti-Sca1 BUV395, anti-CD9-PE, and anti-PDGFRA APC. After washing with FACS buffer cells were sorted. Doublets and dead cells were excluded based on forward and side scatter and DAPI, respectively. Adventitial fibroblasts were identified as Live, Lin- (Dump channel = DAPI, anti-CD45, anti-CD31, anti-EPCAM) and Sca1+. FACS was performed on a BD FACS Aria using FACSDiva Software. ribosomal RNA depletion