GSM8746507: Control 1; Mus musculus; RNA-Seq
Whole upper airway (larynx-trachea) was mechanically dissociated by mincing tissue with razor blades in solution containing 5 ml of RPMI 1640 (Thermo Scientific) with 10% fetal bovine serum, 1 mM HEPES (Life Technology), 1 mM MgCl2 (Life Technology), 1 mM CaCl2 (Sigma), 0.5 mg ml−1 collagenase D and type I/dispase (Roche), and 0.25 mg DNase I (Roche). Minced tissue was then digested by shaking at around 150 rpm for 30 min at 37 °C. Following incubation, upper airway pieces were mechanically dissociated further by straining through a MACS 70 μm filter. Red blood cells were removed by the addition of 1 ml of RBC lysis buffer (BioLegend) to each tube and incubation at room temperature for 1 min. Single-cell suspensions were pelleted (1,500 rpm, 4 °C, 5 min), counted with a hemocytometer and diluted to around 1 × 106 cells ml−1. Diluted cells were stained with Fc blocking antibody (5 mg ml−1, BD). Cells were then incubated with specific surface marker antibody at 1:1000 APC-FITC-conjugated anti-Epcam (BioLegend, no. 118214) to isolate epithelial lineage. Cells were then stained using live/dead dye (CD11b: Brilliant Violet 450, BioLegend, cat#75-0112-U025) before being resuspended in 2% FBS + 1:2000 DAPI. All FACS sorting was done on a BD FACSAria Fusion Sorter (BD Biosciences) analyzer with three lasers (405, 488 and 640 nm) at the Flow Cytometry Core at VA San Diego Health Care System and San Diego Veterans Medical Research Foundation. Single cells were processed into complementary DNA libraries using Chromium Single Cell 3' v3 kit (10X Genomics, Pleasanton, CA) and sequencing was carried out on the NovaSeq (Illumina) platform. The R package Seurat (v4.0)26 was then used to perform data quality control, normalization, principal components analysis, UMAP generation and differential gene expression testing.  Single cells with above 15% mitochondrial reads and between 200 and 7500 unique genes were considered high-quality cells and were filtered for further analyses. Additional filtering included a minimum UMI cutoff of 2 and minimum cell count cutoff of 10. LogNormalize global-scaling normalization method was applied with regressing variables cell cycle, S.Score, G2M.Score, and mitochondrial RNA level to remove confounding effect on downstream clustering. In addition, DoubletFinder (v2.0)64 was used to remove doublets and LogNormalize was again used to normalize feature expression. CCA65 was used to integrate dataset across two experimental batches for a total of n=8 mice (4 males, 4 females).