title: GSM8755902: WT, 2mo SM, 2; Mus musculus; RNA-Seq
library_construction_protocol: left lung lobe from each mouse was further processed to isolate single cell suspensions. Lung tissue was first minced into small pieces and placed in 0.5mL Dispase solution (50U/mL, Sigma) in a conical tube. The tissue was incubated at room temperature on a gentle rocker for 45 minutes. The tissue was then placed in a petri dish with 7mL complete media with 10ml DNase-I solution (1mg/mL, Sigma), teased apart, and placed on a rocker for 12 minutes to liberate adherent cells. Liberated cells were then passed through a 70mm strainer into a conical tube with subsequent washing of the strainer with 5mL of media. This process was repeated with a 40mm strainer. Cells were spun down at 400g for 10minutes at 4 degrees. Cells were resuspended in 6mL of red blood cell lysis buffer, incubated for 5 minutes, and spun down.  Cell pellets were resuspended in 0.5mL PBS with 0.4% bovine serum albumin (BSA, Albuminx from ThermoFisher), filtered a final time through a 40mm FloMi filter tip (Bel-Art), and kept on ice. Cells were counted and 5,000 cells were processed. Individual cells were barcoded with library preparation using 3primeV2 reagents by 10X Genomics Chromium System per the manufacturer's protocol. Next generation RNA sequencing was performed on the libraries using an Illumina NextSeq-500 by the UPMC Genome Center (Pittsburgh, PA).