GSM4518465: Adult murine lung cells; Mus musculus; RNA-Seq
Whole lung was dissected from the adult mouse and 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) and removed from the chest. The lung was further diced with razor blades and the mixture was incubated for 45 mins at 37 °C and vortexed intermittently. The mixture was then washed with FACS buffer (2% FBS in DMEM-F12). The mixture was passed through a 70 µm cell strainer and resuspended in RBC lysis buffer, before passing through a 40 µm cell strainer. Cells suspensions were incubated with the appropriate antibodies in FACS buffer for 30 min at 4 °C and washed with FACS buffer. The following antibodies were used at 1:200 for staining: anti-CD45-PE-Cy7, anti-CD31-Percp, and anti-Epcam-BV421. DAPI (0.2 µg/ml) was used to exclude dead cells. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Cells were sorted into FACS buffer. FACS analysis was performed by FACSDiva. cDNA libraries were prepared for sequencing using standard protocol for the 10x Single Cell instrument, using Single cell 3' v2 chemistry kit. droplet-based single cell RNA-Seq
GEO Accession
GSM4518465