GSM5919612: lung single cell 67; Mus musculus; RNA-Seq
GSM5919612
A 12 week-old female Prox1-GFP mouse was perfused with PBS to clear the blood. Subsequently, the lung was filled with 10 mg mL-1 dispase II in PBS via the trachea and incubated for 10 min at RT. Next, 1.0 mg mL-1 collagenase II in PBS supplemented with 0.2 mg mL-1 DNase I was injected into the lung lobes and lung tissue was minced with scissors in 1.5 mL of 1.0 mg mL-1 collagenase II in PBS supplemented with 0.2 mg mL-1 DNase I. Tissue dissociation was performed for 15 min at 37°C and 750 rpm. Digests were quenched by the addition of 2 mM EDTA f.c., filtered through a 40 µm nylon filter, washed with FACS buffer (PBS, 0.5% FBS, and 2 mM EDTA) for 5 min at 300 g, RT and processed for FACS sorting. Briefly,  Fc-receptor binding was blocked with rat anti-mouse CD16/CD32 for 10 min on ice. Thereafter, samples were incubated with fluorophore-conjugated anti-CD31 (clone 390, 1.0 µg mL-1), anti-CD45 (clone 30-F11, 4 µg mL-1), anti-CD11b (clone M1/70, 4 µg mL-1) and anti-TER-119 (clone TER-119, 4 µg mL-1) antibodies for 20 min on ice. Prior to sorting of CD31+ Prox1-GFP+ CD45- CD11b- Ter119- lymphatic and CD31+ Prox1-GFP- CD45- CD11b- Ter119- blood endothelial cells on a BD FACS Aria III samples were again filtered through a 40 µm filter and supplemented with 1 µM SytoxBlue to label dead cells. Libraries for RNA-seq were prepared by using SMART-seq2 methodology