title: GSM8152115: Lung,KO, WT ,saline; Mus musculus; RNA-Seq
library_construction_protocol: Lungs were perfused with cold sterile saline through the right ventricle. The lungs were then intratracheally inflated with 1.5 ml of Dispase 50 U/ml (Corning) followed by installation of 0.5 ml of 1% low melting agarose (BioRad) via the trachea. The lungs were excised and incubated on an orbital shaker for 45 min at room temperature. Each lobe was then minced into small pieces in DMEM (GIBCO) supplemented with 10% FBS (GIBCO) and 0.33 U/ml DNase I (Roche) and placed on the orbital shaker for an additional 10 minutes. The cells were then filtered through a 100 µm cell strainer and centrifuged at 400 g for 5 min at 4°C. The cell pellet was resuspended in ACK-buffer (GIBCO), incubated for 2 min on ice and then filtered through a 40 µm cell strainer. The cells were centrifuged at 400 g for 5 min at 4°C and resuspended in DPBS (GIBCO) supplemented with 5% FBS, and then stained with the following antibodies: EpCAM-BV785 (BioLegend #118245), CD45-APC (BioLegend, 103112), CD31-APC/Cy7 (BioLegend, 102534), I-A/I-E - AlexaFluor488 (MHC-II) (BioLegend, 107616) and 4', 6-diamidino-2-phenylindole (DAPI) (Life Technologies, 62248) was used to eliminate dead cells. The cells were then sorted on the BD FACS Aria III system (BD Bioscience). Single cell capturing was performed using the BD Rhapsody™ Express Single-Cell Analysis System (BD Biosciences). Libraries were generated using BD Rhapsody Whole Transcriptome Analysis (WTA) Amplification kit according to the manufacturer's protocols.