LC-MS/MS quantification of m6A in total RNA

Total RNA was isolated using TRIzol reagent (Invitrogen, USA) following to the manufacturer’s instruction. 1 μg of total RNA was digested by 4 μl nuclease P1 (Sigma, USA) in 40 μl buffer solution (10 mM Tris-HCl pH 7.0, 100 mM NaCl, 2.5 mM ZnCl2) at 37 °C for 12 h, followed by incubating with 1 μl alkaline phosphatase (Sigma, USA) at 37 °C for 2 h. RNA solution was diluted to 100 μl and injected into LC-MS/MS. The nucleosides were separated by reverse phase high-performance liquid chromatography on an Agilent C18 column, coupled with mass spectrometry detection using AB SCIEX QTRAP 5500. The m6A levels were calculated as the ratio of m6A to A based on the calibrated concentrations according to the standard curve obtained from pure nucleoside standards running with the same batch of samples.