GSM5733161: Old_GFP-pos_p16INK4a+ 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. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence and GFP+ 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.