Quantifying m6A Levels in Cellular RNA

The polyadenylated RNA from transfected cells was isolated using a biotinylated poly(dT) probe followed by a rRNA depletion step to ensure depletion of rRNA (Supplementary Fig. 1e). The isolated RNA was subsequently digested into nucleosides and the amount of m⁶A was measured by LC-MS/MS following the published procedure^{5 (link)}; the total contents of m⁶A and A were quantified based on the corresponding standard curves generated using pure standards (Supplementary Fig. 2), from which the m⁶A/A ratio was calculated. Typically, 200–300 ng of polyadenylated RNA was digested by nuclease P1 (2 U) in 25 μL of buffer containing 25 mM NaCl, and 2.5 mM ZnCl₂ at 37 °C for 2 h, followed by additions of NH₄HCO₃ (1 M, 3 μL) and alkaline phosphatase (0.5 U) and incubation at 37 °C for 2 h. The sample was then filtered (0.22 μm pore size, 4 mm diameter, Millipore), and 5 μL of the solution was injected into LC-MS/MS. The nucleosides were separated by reverse phase ultra-performance liquid chromatography on a C18 column with online mass spectrometry detection using Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified by using the nucleoside-to-base ion mass transitions of 285 to 153 (d₃-m⁶A), 282 to 150 (m⁶A), and 268 to 136 (A). Quantification was performed in comparison with the standard curve obtained from pure nucleoside standards running on the same batch of samples. The ratio of m⁶A to A was calculated based on the calibrated concentrations.