m6A RNA immunoprecipitation (meRIP) and RT-qPCR

Total RNA from three biological replicates of MCF-7 cells was isolated with TRIzol (15596018, Invitrogen) according to the manufacturer's instructions. RNA was diluted to 1 µg/µL and fragmented with RNA Fragmentation Reagents (AM8740, Invitrogen) at 70°C for 5 min. A small aliquot of fragmented RNA (500 ng) was reserved in 10 µL nuclease-free water for use as the input sample in qRT-PCR normalization. Protein A/G Magnetic Beads (88803, Pierce) were washed twice with IP Buffer (20 mM Tris pH 7.5, 140 mM sodium chloride, 1% NP-40, 2 mM EDTA) and coupled with anti-m⁶A antibody (ab151230, Abcam) or an IgG control (NB810-56910, Novus) for 1 h at room temperature. The beads were washed three times with IP Buffer, then 10 µg fragmented RNA and 400U RNase inhibitor was added to 1 mL IP Buffer. The antibody-coupled beads were resuspended in 500 µL RNA mixture and incubated 2 h to overnight at 4°C on a rotor. The beads were then washed three times with cold IP Buffer. Samples were eluted with 200 µL Elution Buffer (1× IP Buffer containing 10 U/µL RNase inhibitor and 0.5 mg/mL N⁶-methyladenosine 5′-monophosphate [M2780, Sigma-Aldrich]) for 2 h at 4°C on a rotor. The supernatant was removed, and ethanol precipitated with 2.5 M Ammonium acetate, 70% Ethanol, and 50 µg/mL GlycoBlue Coprecipitant (Invitrogen AM9515). The RNA was washed with 70% ethanol, dried for 10 min at room temperature, and then resuspended in 10 µL nuclease-free water. The RNA was reverse transcribed using SuperScript IV Reverse Transcriptase (18090010, Invitrogen) and quantified by qPCR using primers specific to an identified m⁶A site or region with no m⁶A in the same gene. Percent input calculation was performed based on the resulting Cq values.