N6 methyladenosine m6a antibody

m6A-modified TFRC was pulled down with N6-methyladenosine (m6A) antibody (Active Motif, China) for m6A-RIP. We used mRNA Isolation Kit (NEB, China) to extract and purify total RNA of HCT-116 cells. Then we prepared the IP buffers as literature described [31 ]. Protein A/G PLUS-Sepharose beads were used to antibody-immobilized (m6A antibody or IgG) in the IP buffer. The purified RNA was then transferred into EP tubes containing RNase and protease inhibitors and incubated overnight at 4℃. Finally, qRT-PCR analysis was performed after RNA extraction. Table S2 lists the primers used in this assay.

For m6A-RNA immunoprecipitation, we pulled down m6A-modified SCD using an N6-methyladenosine (m6A) antibody (Active Motif, China). A polyA Spin mRNA Isolation Kit (NEB, China) purified total RNA extractive from Hela cells after treatment. Protein A/G PLUS-Agarose beads were incubated (m6A antibody and IgG) by IP buffer as described above [39 (link)]. Then the depurated RNA was put in the aforesaid EP tube including proteinase and RNase inhibitor, then hatched at 4 °C through a night. We made qRT-PCR analysis after RNA extracted by an RNA extraction kit, using the primers presented in Table S2. The IP assay embodied the protein expression of m6A. In short, we added 100 µl of the antibody-immobilized beads-RNP complex to a fresh EP tube, tackled using SDS–PAGE, and performed western blot.

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.