GSM9534882: Pou2f3 KO Infected- PR8 - Day 22; Mus musculus; RNA-Seq
Lungs were harvested from mice and single-cell suspensions were prepared. Briefly, lungs were perfused with ice cold PBS via the left atrium, lobes were separated and collected in ice cold Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific) +10% fetal bovine serum (FBS; Thermo Fisher Scientific).  Once all mice were harvested, the lobes were placed in 1.5 mL of digestive media (DMEM with 0.15 mg/ml of Liberase TM (Roche # 5401119001),  0.014 mg/mL of DNase I (Sigma Aldrich # D4527-20KU) and 0.20 mg/mL of Dispase II (Sigma Aldrich D4693-1G), cut into small pieces and placed at 37°C to shake for 40-mins. The samples where then passed through a 16-G needle three times and placed back shaking at 37°C for 15-mins. The samples were then passed through a 18-G needle three times and filtered through a 70 µm strainer. The filters were washed with DMEM+10% FBS. Red blood cells were then lysed for 3-mins (Thermo Fisher Scientific #A1049201) and cells were manually counted using a hemacytometer. Whole-lung single-cell suspensions were then used for subsequent experiments Single-cell RNA-Seq was performed using the Chromium System (10x Genomics) and the Chromium Single Cell 3' Reagent Kits v2 (10x Genomics) at the Children's Hospital of Philadelphia Center for Applied Genomics.