Quantification of Methylated RNA

Methylated RNA was purified using the RNA Clean & Concentrator Kit (Zymo Research). The purified RNA was subsequently enzymatically digested to individual mononucleosides using nuclease P1 (Sigma Aldrich), snake venom phosphodiesterase (Sigma Aldrich), and Calf Intestinal Phosphatase (NEB) [2 (link)–4 (link),20 (link),51 (link)]. The digested samples were separated by analytical HPLC using a Luna C18 reverse-phase column (10 μm, 4.6 mm x 250 mm) (Phenomenex) and as in a previously described protocol [20 (link)]. Synthetic 2-methyladenosine and the unmodified mononucleosides were detected by UV absorption at 256 nm, whereas the ¹⁴C-labeled methyladenosines were detected by radiomatic flow scintillation analyzer (Packard 515TR flow scintillation analyzer; Perkin-Elmer).