title: GSM4176344: GFPneg time 0 rep 3; Mus musculus; RNA-Seq
library_construction_protocol: 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 ºC 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 ºC and washed with FACS buffer. 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. CD45-Epcam-CD31-PDGFRa+GFP- or CD45-Epcam-CD31-PDGFRa+GFP+ cells were sorted . Cells were sorted into FACS buffer. FACS analysis was performed by FACSDiva (BD Biosciences). The RNA was extracted by PicoPure RNA Isolation Kit (Applied Biosystems) from the sorted cells, and the amount and quality of extracted RNA was measured by RNA 6000 Pico Kit (Agilent). The Bulk RNA-seq was done in GENEWIZ. RNA libraries were prepared for sequencing using standard Illumina protocols
identifiers.namespace: GEO
identifiers.id: GSM4176344