GSM7324826: Old_VPF_replicate_1; Mus musculus; RNA-Seq
Samples pooled from 2 mice per group included 25 mg of lung tissue from each mouse, making a total 50 mg used for the subsequent tissue dissociation. Each pool of 50 mg of lung tissue was placed into a Miltenyi C tube filled with 2 ml of a mixture of RPMI media, 0.2 mg/ml Liberase TL, and 0.1 mg/ml Elastase. The following program was then run in the Gentlemacs Octodissociator (Miltenyi) at a constant temperature of 37C: 20 min, 50 rpm; 30 sec, 2000 rpm (clockwise); 30 sec, 2000 rpm (counterclockwise). Samples were afterwards passed through a MACS SmartStrainer (70 µm), centrifuged, and the remaining pellets were resuspended in RPMI + 20% FBS. Following collection and dissosiation of the mouse lungs, cells were first incubated for 5 min with 5 µl of TruStain FcX™ PLUS (anti-mouse CD16/32) blocking reagent followed by a 15-min incubation with 5 μl of anti-mouse Ter119-Pacific Blue and 5 μl of anti-mouse CD45-PE/Cyanine7 for 15 min at 25°C. Cells were washed with 1 ml of DMEM supplemented with 15% FBS and then resuspended in 500 μl of DMEM supplemented with 15% FBS, 2 μg/ml of propidium iodide, and 50 mM EDTA. Lastly, 25,000 cells were sorted using FACS Aria FUSION (BD Bioscience). Single-cell libraries were constructed using the Chromium Next GEM Single Cell 3' Kit v3.1 with Chromium Next GEM Chip G Single Cell Kit in a Chromium Controller (10x Genomics) according to manufacturer's protocol.