Total RNA extraction was conducted by using the RNAiso Plus (Takara, Shiga, Japan), after which DNase was added to remove DNA. An m6A RNA enrichment kit (EpigenTek) was used to detect MeRIP levels according to the manufacturer’s instructions. The m6A-containing target fragment was pulled down using a bead-bound m6A capture Ab, after which the RNA sequence containing both ends of the m6A region was cleaved using a lyase cocktail. The enriched RNA was released, purified, and eluted. Quantitative real-time PCR (QRT-PCR) was performed after MeRIP to quantify changes in target gene m6A methylation.
Antibodies against ALKBH5 (Abcam, #ab195377) were used in RNA-binding protein immunoprecipitation (RIP) assays. RIP assays were performed using an EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Cells at approximately 90% confluence were lysed using complete RIP lysis buffer containing RNase Inhibitor (Millipore) and protease inhibitor, and 100 μl of whole-cell extract was subsequently incubated with RIP buffer containing magnetic beads conjugated to specific antibodies. The detailed method of the RIP assay was described in a previously published study12 (link).
Antibodies against ALKBH5 (Abcam, #ab195377) were used in RNA-binding protein immunoprecipitation (RIP) assays. RIP assays were performed using an EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Cells at approximately 90% confluence were lysed using complete RIP lysis buffer containing RNase Inhibitor (Millipore) and protease inhibitor, and 100 μl of whole-cell extract was subsequently incubated with RIP buffer containing magnetic beads conjugated to specific antibodies. The detailed method of the RIP assay was described in a previously published study12 (link).
Free full text: Click here
