GSM9040799: scATAC_HDM-only; Mus musculus; ATAC-seq
After being dissected in PBS and cut into pieces using forceps, the entire lung was digested in 1 ml Liebovitz media (Gibco, 21083-027) for 30 minutes at 37°C with 0.5 mg/mL of DNase I (Worthington, D, LS002007), 2 mg/mL of collagenase type I (Worthington, CLS-1, LS004197), and 2 mg/mL of elastase (Worthington, ESL, LS002294). By pipetting up and down, a mechanical trituration was performed on the tissue at 15 minutes during the digestion until the piece were small enough to go through pipet tip. Following the addition of 20% fetal bovine serum, 300 µl (FBS, Invitrogen, 10082–139) to stop the enzymatic reaction, the tissue was homogenized by trituration. The samples were put on ice in the cold room, filtered through a 70 μm cell strainer (Falcon, 352350), and spun down for one minute at 5000 rpm. This was followed by the removal of the supernatant, the addition of 1 ml of red blood cell lysis buffer (15 mM NH₄Cl, 12 mM NaHCO₃, 0.1 mM EDTA, pH 8.0), and the samples' incubation on ice for 3 minutes. Cells were pelleted again by centrifugation at 5000 g for 1 min, washed with once Liebovitz + 10% FBS, resuspended with 1 ml Liebovitz + 10% FBS and filtered through 70 μm cell strainer into a 5 ml glass tube. Samples 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 on ice. SYTOX Blue (1:1000, Invitrogen, S34857) was added for viability. A BD FACS Aria Fusion Cell Sorter was used to sort the samples. Dead cells and doublets were excluded. For scRNA-seq and scATAC-seq, CD45-positive cells were collected as the immune cell lineage; from the CD45-negative population, ICAM2-positive cells were collected as the endothelial cell lineage; from the ICAM2-negative population, ECAD-positive cells were collected as the epithelial cell lineage; and the remaining triple-negative population was collected as the mesenchymal lineage. Epithelial cell lineage was submitted for the 10x Genomics library. Lung epithelial cells and immune cells (CD45+) were sorted from 8-week-old mice and processed using the 10x Genomics Chromium Single Cell ATAC platform with the 3′ Library and Gel Bead Kit, following the manufacturer's protocol.