GSM8477271: Scgb1a1CreERT- control; Mus musculus; RNA-Seq
Lung tissues were harvested from mice and incubated with digestion media (RPMI, 2mg/ml collagenase A, and 0.1mg/ml DNaseI) at 37°C for 20 minutes on a rocker. The resulted single cell suspension was filtered through a 70um filter, then subsequently incubated with ACK lysis buffer for 3 minutes. Cells were stained with viability dye and were resuspend in FACS staining buffer (PBS, 2% FBS, 0.1% sodium azide). Cells were then stained with conjugated anti-CD45 antibody for 30 minutes at 4°C. Cells then were sorted to live CD45+ and CD45- populations. Then the two populations were mixed at 1 to 1 ratio for scRNA-seq. Enzyme fragmentation, A tailing, adaptor ligation and PCR are performed to obtain final libraries containing P5 and P7 primers used in Illumina bridge amplification. Size distribution and molarity of resulting cDNA libraries were assessed via Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies, USA).