GSM9067549: Ctrl, replicate 1, scRNA; Mus musculus; RNA-Seq
The tracheal epithelial cells were isolated from 2 control mice (Mettl3fl/+; Krt5-CreERT2/+; R26R-tdTomato) and 2 Mettl3 conditional knockout mice (Mettl3fl/fl; Krt5-CreERT2/+; R26R-tdTomato) by dissecting tracheas, removing connective tissue, and mincing into small pieces followed by sequential enzymatic digestion in DMEM/F12 containing 2 mg/mL dispase (Sigma, D4693) at 37°C for 30 minutes and 0.5 mg/mL DNase I (Solarbio, D8071) at 37°C for 30 minutes, after which epithelial sheets were manually peeled and further dissociated into single cells using 0.25% Trypsin-EDTA (Thermo Fisher, 25200056) at 37°C for 5 minutes, with the resulting cell suspension filtered through a 70 μm strainer before preparing a 1:1 mixture of Krt5-lineage-traced tdTomato+ and non-traced tdTomato- cells for 3' droplet-based single-cell RNA sequencing (10x Genomics platform). The cell suspension was loaded into 10x Chromium Chip (v3.1) and barcoded with a 10x Chromium Controller. RNA from the barcoded cells was subsequently reverse-transcribed, amplified and prepared into sequencing libraries with 10x Library Construction Kit (v3.1) according to the manufacturer's instructions.