GSM9888082: Supernatant_1; Mus musculus; OTHER
EpCAM⁺/CD45⁻ epithelial cells were isolated from wildtype mouse esophagus via single-cell dissociation and magnetic-activated cell sorting (MACS) enrichment. Total RNA was extracted  with miRNeasy Micro (Qiagen) according to the manufacturer's instructions, with a portion retained as Input and the remainder subjected to m6A immunoprecipitation. Anti-m6A antibody (1.25 µg/reaction) was pre-bound to Protein A/G magnetic beads (Millipore) for 1 h, then incubated with RNA at 4 °C for 2 h. Beads were sequentially washed (low salt ×2, high salt ×2, RIPA ×2), with washes collected as Unbound. m6A-bound RNA was specifically eluted using 20 mM m6A 5′-monophosphate sodium salt at 4°C for 1 h, followed by ethanol precipitation. Libraries for Input, Unbound, and m6A-bound fractions were prepared using NEBNext Single Cell/Low Input RNA Library Prep and sequenced on Illumina HiSeq 2500 (PE150) with three biological replicates per fraction.