2.3. RNA Digestion and LC-ESI-MS/MS Analysis

In total, 500 ng of RNA samples was denatured for 15 min at 95 °C and fully digested by S1 nuclease (180U, Promega, Madison, WI, USA) for 4 h at 37 °C. After the addition of alkaline phosphatase (CIAP, 30U, Promega, Madison, WI, USA) and Venom phosphodiesterase I type IV (0.01U, Sigma-Aldrich, Saint Louis, MO, USA), the samples were incubated for another 1 h at 37 °C. The digested RNA samples were diluted in 100 µL Mili-Q water (final volume 200 µL) and extracted twice by equal volume of chloroform. Afterward, the resulting aqueous layers were collected, lyophilized to dryness, and redissolved in 100 µL Mili-Q water. LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometric) analysis of nucleosides was performed on a Shimadzu LCMS8060 mass spectrometer (Shimadzu, Japan) connected to a Nexera Mikros HPLC system (Shimadzu, Japan). Separation was carried out on a Waters NanoACQUITY Symmetry C18 column, 150 × 0.3 mm, 3.5 µm particle size. As mobile phases, formic acid in water (0.1%, v/v) and formic acid in methanol (0.1%, v/v) were used (phases A and B, respectively). Elution started with 5% solvent B for 5 min, then was raised to 30% solvent B in 10 min and to 50% solvent B in 5 min; after 3 min, the equilibration solvent ratio was set back to 5% solvent B for another 10 min. The flow rate was 5 µL/min, and the column temperature was 30 °C. The MS detection was performed under positive electrospray ionization mode, and the source parameters were as follows: probe voltage 2.6 kV, desolvation line temperature 250 °C, heater block 400 °C, nebulizing gas 1L/min. The triplequad analyzer was operated in MRM mode, and the specific reaction for m⁶A fragmentation was monitored (282.1→150.1), as well as for A (268.1→136.1), which was used for reference. All mass chromatograms were analyzed employing Shimadzu Lab Solutions software. Quantitation was based on calibration curves, which were constructed by plotting the mean peak area ratio of m⁶A/A versus the mean molar ratio of m⁶A/A (ranging from 0.05 to 5% of m⁶A/A) (Figure S1).