Anti m6a antibody

For quantification of m⁶A-modified CYR61 levels, methylated RNA immunoprecipitation was performed. Total RNA was isolated from HTR-8 cells by Trizol. 3 μg of anti-m⁶A antibody (Millipore, ABE572) or anti-IgG (Cell Signaling Technology) was conjugated to protein A/G magnetic beads in IP buffer (20 mM Tris pH 7.5, 140 mM NaCl, 1% NP-40, 2 mM EDTA) for overnight at 4 ^oC. A 100 μg aliquot of total RNA was then incubated with the antibody in IP buffer supplemented with RNase inhibitor and protease inhibitor. RNA was eluted from the beads by incubating with 200 μl 0.5 mg/mL N⁶-methyladenosine 5-monophosphate sodium salt (Sigma-Aldrich) for 1 h at 4 ºC. Total RNA was eluted with elution buffer, purified through phenol-chloroform extraction. For further qRT-PCR assay, 10 ng of eluate or input total RNA was reverse-transcribed using Superscript III with random hexamers, and enrichment of m⁶A-containing transcripts. Fold enrichment was calculated by calculating the 2^-ΔCt of eluate relative to the input sample. The primers used for PCR were as follows: CYR61 primer for MeRIP-qPCR #1 F: 5'-GAATGCAGCAAGACCAAGAAAT-3', R: 5'-ACGCAGTACTTGGGCCGGTAT-3'; CYR61 primer for MeRIP-qPCR #2 F: 5'-TTTCCAAGAACGTCATGATGAT-3', R: 5'-CCTGGAAACC- CAGGTAGCAT-3'.

Total RNA was separated from pre-processed cells and fragmented into 100 nucleotides at random. Then, RNA samples were immunoprecipitated with magnetic beads pre-coated with anti-m6A antibody (Millipore, Germany) or anti-mouse IgG (Millipore). Next, m6A modified RNA fragments were eluted with N6-methyladenosine 5′- sodium monophosphate. Finally, qPCR detection was performed.

MeRIP-qPCR was performed according to the published literature with slight modifications. In brief, total RNA (150 μg) was isolated from pretreated cells (SiHa) and randomly fragmented into 100-nt RNA fragments using ZnCl2. RNA samples were immunoprecipitated with antibodies (10 μg anti-m⁶A antibody or anti-mouse immunoglobulin G [IgG]; Millipore, Germany) pre-coated with magnetic beads to elute the m⁶A-modified RNA fragments from the beads with m⁶A in IP buffer (B5993; ApexBio Technology). MeRIP-qPCR was performed using the Magna MeRIP m⁶A Kit (Millipore, Germany) to detect the m⁶A modification quantitation. The RNA enrichment was analyzed by quantitative real-time RT-PCR.

m⁶A modification of ZFAS1 was detected using Magna MeRIP™ m⁶A Kit (Millipore) as the manufacturer’s instructions. Briefly, 24 h after transfection, cells were harvested to perform RIP experiments using an m⁶A antibody. 1.5 μg anti-m⁶A antibody (Millipore) or anti-IgG (Cell Signaling Technology) was conjugated to protein A/G magnetic beads overnight at 4°C. A total of 100 μg RNA was then incubated with the antibody in IP buffer. Then, the purified RNA was analyzed via RT-PCR assay.