GSM9140147: Ctrl, replicate 3, RNA-seq; Mus musculus; RNA-Seq
tracheal from Ctrl and M3-CKO mice were dissected, cleaned of connective tissue, and minced into pieces. The tissue were then incubated in DMEM/F12 medium containing 2mg/mL dispase (Sigma; D4693) at 37°C for 30 minutes. Subsequently, tracheas were incubated in 0.5mg/mL DNase I (Solarbio; D8071) for additional 30 minutes at 37°C. The epithelial sheets were delicately peeled using forceps and further digested in 0.25% Trypsin-EDTA (Thermo Fisher Scientific; 25200056) at 37°C for 5 minutes to obtain single cells. The resulting cells suspension was gently pipetted and passed through a 70 μm cell strainer to removed any remaining tissue fragments. Cells were buried in metrigel and culture immediately in mouse tracheal epithelial cell culture medium for 14 day. On day 14, organoids were harvested for  RNA extraction. Total RNA was extracted using Trizol reagent. Briefly, purified mRNAs were fragmented using an RNA fragmentation kit. A portion of the fragmented mRNA (~4%) was reserved as input control, while the remaining fragmented RNA was incubated with an anti-m6A antibody and subjected to m6A immunoprecipitation (IP) using the EpiMark N6-Methyladenosine Enrichment Kit according to the manufacturer's protocol. Both the IP-purified fragmented mRNA and input RNA were used to construct libraries with the SMARTer Stranded Total RNA-seq Kit v2-Pico Input Mammalian, following the manufacturer's instructions.