GSM7500884: 2042GFPPOS; Mus musculus; RNA-Seq
Dissected mouse lung was tracheally perfused 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) after perfusion with PBS and removed from the chest. The lung was incubated in digestion cocktail for 45 mins at 37C with continuous shaking. The mixture was then washed with FACS buffer (2% FBS and 1% Penicillin-Streptomycin in DMEM). The mixture was passed through a 70 μm cell strainer and resuspended in red blood cell (RBC) lysis buffer, then passed through a 40 μm cell strainer. Cell suspensions were incubated with the appropriate conjugated antibodies in sorting buffer for 30 min at 4C and washed with FACS buffer. Doublets and dead cells were excluded based on forward and side scatter and SYTOX Blue (Invitrogen, S34857), respectively.  The following antibodies were used for staining: CD45-BV421 (BD, 563890), CD31-BV421 (Invitrogen, 48-0311-82), EpCAM-BV421 (BD, 563214). Immune (CD45-biotin, Biolgened, Cat#103104), epithelial (CD326-biotin, Biolegend, Cat#118204) and endothelial (CD31-Biotin, Biolegend, Cat#102404) cells are removed with EasySep mouse streptavidin RapidShperes (StemCell, 19860A). FACS was performed on a BD FACS Aria using FACSDiva Software. CD45- CD31- EpCAM- cells were sorted for mesenchymal cells, the GFP+ fibroblasts were further separated and were sorted into FACS buffer. cDNA libraries were prepared for sequencing using standard protocol for the 10x Single Cell instrument.