M6A Enrichment and Analysis Protocol

For Me-RIP, an N6-methyladenosine (m⁶A) antibody (Active Motif, China) was used to pull down m⁶A-modified MYC. Total RNA was extracted from treated SiHa cells and purified using a polyA Spin mRNA Isolation Kit (NEB, China). The IP buffer was prepared as previously described 20 (link). Antibody-immobilized Protein A/G PLUS-Agarose beads were prepared by incubating with m⁶A antibody and IgG antibody in IP buffer. The purified RNA was then added to the above-mentioned microcentrifuge tubes containing protease and RNase inhibitors followed by incubation at 4 °C overnight. The RNA was extracted using an RNA extraction kit and analyzed by qRT-PCR, the primers used for qPCR are listed in Table S2. The protein expression of m⁶A was detected by IP assay. Briefly, 100 µl of the mixture containing the antibody-immobilized beads-RNP complex was added to a new tube, resolved by SDS-PAGE, and subjected to western blotting.