GSM3132037: T_Tr_2; Mus musculus; RNA-Seq
For tracheal epithelium, the trachea was dissected from mice, stored in DMEM + 5% FBS and cleaned of stroma using a dissecting microscope. Tissue was cut into 6 pieces and incubated at room temperature in 16U mL-1 Dispase (Corning) in DPBS without Ca2+/Mg2+. Incubation was monitored carefully and stopped by transfer into DMEM + 5% FBS when epithelium began to lift away from stroma (23-28 minutes). Epithelium was then peeled off under a dissecting microscope and collected in DMEM + 5% FBS on ice. Harvested epithelium was digested in 0.5% Trypsin + EDTA for 20 minutes at 37° C. After digest, cells were vortexed briefly and trypsin was quenched with DMEM + 5% FBS. Cells were washed once and stained for flow cytometry. Cells were sorted directly into lysis buffer from the Dynabead mRNA Direct Purification Kit (ThermoFisher). Sorted cells were pooled for bulk sequencing and poly A RNA was isolated according to the manufacturer's protocol. amplification protocol: Amplified cDNA was prepared using the NuGen Ovation RNA-Seq system V2 kit, according to the manufacturer's protocol Sequencing libraries were generated using the Nextera XT library preparation kit with multiplexing primers, according to manufacturer's protocol (Illumina)