GSM8242374: Control scRNAseq_Car4; Mus musculus; RNA-Seq
Perfused lungs were collected from anesthetized mice as described above. Previously published protocols for cell dissociation and sorting cells were used with minor modifications11. Extra-pulmonary airways and connective tissues were removed from lungs, which were then minced with forceps and digested in Liebovitz media (Gibco, 21083-027) for scRNA-seq, ATAC-seq, or scATAC-seq with 2 mg/mL collagenase type I (Worthington, CLS-1, LS004197), 0.5 mg/mL DNase I (Worthington, D, LS002007), and 2 mg/mL elastase (Worthingon, ESL, LS002294) for 30 min at 37 °C. To stop the digestion, fetal bovine serum (FBS, Invitrogen, 10082-139) was added to a final concentration of 20%. Tissues were triturated until homogenous and filtered through a 70 µm cell strainer (Falcon, 352350) on ice in a 4 °C cold room and transferred to a 2 mL tube. The sample was then centrifuged at 1,537 rcf for 1 min; then 1 mL red blood cell lysis buffer (15 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA, pH 8.0) was added and incubated for 3 min before centrifugation again at 1,537 rcf. The red blood cell lysis buffer incubation was repeated to remove residual red blood cells. Liebovitz+10% FBS was used to wash and resuspend samples, which were then filtrated through a 35 mm cell strainer into a 5 mL glass tube and had SYTOX Blue (1:1000, Invitrogen, S34857) added. After refiltering, samples were for ATAC-seq were sorted for GFP positive cells from a RosaSun1GFP reporter activated by Wnt3aCre, Rtkn2CreER, or SftpcCreER on a BD FACSAria IIIu cell sorter with a 70 mm nozzle. Samples for scRNA-seq or scATAC-seq were stained with CD45-PE/Cy7 (BioLegend, 103114), ECAD-PE (BioLegend, 147304), and ICAM2-A647 (Invitrogen, A15452) antibodies (1:250 dilutions for all antibodies) for 30 min followed by a wash and being resuspended with Liebovitz meda+10% FBS and addition of SYTOX Blue. Samples were then filtered through a 35 mm cell strainer into a 5 mL glass tube again and sorted by a BD FACSAria Fusion sorter or a with a 70 mm nozzle. FACS-purified FACS-purified lung epithelial, immune, endothelial, and mesenchymal c, were processed using the 3′ Library and Gel Bead Kit following the manufacturer's users guide (v2 rev D) on the Chromium Single-Cell Gene Expression Solution Platform (10X Genomics). Library was performed according to the manufacter's instructions (single cell 3' v3 protocol)