Quantification of RNA Modifications

Chromatographic grade acetonitrile was bought from Merck KGaA (Darmstadt, Germany). Methanol of HPLC grade was purchased from J.T.Baker (Radnor, PA, United States). Formic acid (HCOOH) was bought from Fluka (Muskegon, United States). Ammonium formate, malic acid and 5-methylcytidine (m⁵C) were bought from Sigma-Aldrich (St Louis, MO, United States). 2′-O-methyladenosine (Am), N⁶-methyladenosine (m⁶A), 2′-O-methylguanosine (Gm), 1-methylguanosine (m¹G), 2′-O-methylcytidine (Cm), 2′-O-methyluridine (Um), 5-methyluridine (m⁵U) and isotopically labeled standards [D₃]m⁶A, [D₃]Um and [¹³C₅]m⁵U were obtained from Toronto Research Chemicals (Toronto, Canada). [¹³C¹⁵N₂]Gm, [¹³C¹⁵N₂]m¹G and [¹³C₅]Cm were synthesized according to literature (Fu et al., 2015 (link)). Water was purified by a Milli-Q water purification device (Millipore, Milford, MA, United States).