Adenosine

Acetonitrile of chromatographic grade was purchased from Merck KGaA (Darmstadt, Germany). A solution of formic acid (HCOOH) was purchased from Fluka (Muskegon, USA). Ammonium formate and malic acid were obtained from Sigma-Aldrich (St Louis, MO, United States). Adenosine, N⁶-methylAdenosine, N¹-methylAdenosine, 2′-O-methylguanosine (G_m), N¹-methylguanosine, N²-methylguanosine (m²G), 2′-O-methylcytidine, uridine, 3-methyluridine (m³U), 5-methyluridine (m⁵U), and 2′-O-methyluridine and isotopically labeled standards, including [¹³C₅] A, [D₃] m⁶A, [D₃] m¹A, [D₃] C_m, [¹³CD₃] m⁵C, [¹³C₅N₂] U, [D₃] U_m, [¹³C₅] m⁵U, and [D₆] m^2,2G, were purchased from Toronto Research Chemicals (Toronto, Canada). In order to purify water, a Milli-Q water purification device was used (Millipore, Milford, MA, United States).

Isolation of U2 snRNA or RP11-390F4.3 from cells for UPLC-ESI-MS/MS analysis was performed as previously described [58 (link)]. Briefly, the synthetic biotin-labeled probes against specific RNAs were incubated with total RNA and then purified using streptavidin T1 beads. The biotin-labeled probes used are listed in Additional file 2: Table S14. Five hundred nanograms of U2 snRNA or RP11-390F4.3 were treated with 2 units of nuclease P1 (Sigma-Aldrich) at 37°C in 50 μl of reaction buffer (20 mM sodium acetate [pH 5.3], 5 mM ZnCl₂, and 50 mM NaCl). After 4 h, 10 units of antarctic phosphatase (NEB) were added, and the reaction continued for another 4 h. The resulting nucleosides were analyzed by UPLC-ESI-MS/MS with the same settings used to determine 6mA. Nucleoside standards (Toronto Research Chemicals), adenosine, and N⁶,2′-O-dimethyladenosine (m⁶Am) were used to construct daughter ion scan spectra to identify digested RNA products and for quantitative calculation by multiple reaction monitoring (MRM). The nucleosides of the digested RNAs were identified using LC retention with MS/MS spectra and quantified based on the ion mass transitions; m⁶Am (m/z): 296 to 150 and adenosine (m/z): 268 to 136 in MRM mode.