GSM7890847: Expt2_12w hom RFP r1; Mus musculus; RNA-Seq
Lungs were cleared of blood through perfusion and inflated with 2 mL of dispase solution via intratracheal injection and incubated on ice for 2 min. Individual lobes were separated from each lung and minced into small pieces (<1 mm in size). Cells were  washed down to the bottom of the tube using 3 mL of cold PBS and 60 μL of 100 mg/mL collagenase/dispase solution was added per tube. Samples were then placed in a shaking incubator set to 180 rpm at 37 °C  for 45 mins with 7.5 μL of 1% DNase I added to each sample in the final 10 min of incubation. Dissociated tissue cell suspensions were filtered sequentially through 100 μm and 40 μm cell strainers respectively and washed at each stage with 3 mL of 10% FBS in PBS (PF10) after each step to collect remaining cells. Tubes were then centrifuged at 800 rpm for 5 min at 4 °C, after which the supernatant was aspirated. Cell pellets were resuspended in 1 mL of red blood cell lysis buffer. Following a 90 second incubation at RT, cell lysis was inactivated with addition of 6 mL of DMEM/F-12, with 500 μL of FBS then added slowly to the bottom of each tube to collect live cells. After a second centrifugation step at 800 rpm for 5 min at 4 °C, the supernatant was aspirated, and the cell pellets were resuspended in PF10 and placed into separate Eppendorf tubes ready for FACS antibody staining, with a small fraction of cells set aside for unstained and single-stained antibody controls. EpCAM+RFP+ and EpCAM+YFP+ cells were isolated and submitted for library preparation and 10x Genomics sequencing. For each run, libraries were multiplexed and sequenced in the same lanes using NovaSeq 6000. Library construction was performed according to manufacturers instructions (single cell 3' v2 chemstry, 10x Genomics)