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LC–MS/MS for determination of RNA modifications
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RNA ac4C and m6A quantification by LC–MS/MS was performed as described previously25,68,69. In brief, 200 ng of total RNA or other RNA components isolated from it (for example, poly(A) RNA, rRNA and tRNA) were digested by nuclease P1 (1 U, New England Biolabs) at 37 °C for 2 h, followed by the addition of alkaline phosphatase (1 U, Takara) and incubation at 37 °C for another 2 h. The nucleosides were separated by reverse-phase ultra-performance liquid chromatography on a C18 column (Agilent) with online MS detection using Agilent 6470 Triple Quadrupole LC–MS System at a flow rate of 0.25 ml min−1. Buffer A: 0.1% formic acid; buffer B: 50% acetonitrile, 0.1% formic acid with the gradient as follows: 0–1 min, 100% A; 1–2.4 min, 99.8% A; 2.4–3.8 min, 99.2% A; and 3.8–5.2 min, 98.2% A. Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards. The ratio of ac4C to C or m6A to A was calculated based on the calculated concentrations.
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