tRNA isolation and LC-MS analysis

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instruction, with an additional DNase I treatment to eliminate DNA contamination. The RNA was then purified using a RNA Clean & Concentrater^TM -5 kit (R1016, Zymo Research).

tRNA was isolated from the total RNA by gel electrophoresis using 7.5% PAGE (29:1 acrylamide:bisacrylamide) containing 7 M urea. Bands of 60–90 nt tRNA were cut from the gel, extracted using 0.3 M NH₄Ac, and precipitated with glycogen and ethanol. The purified tRNA was then hydrolysed to single nucleosides and dephosphorylated in a 50-μl reaction containing 10 U benzonase (Sigma), 0.1 U phosphodiesterase I (US Biological), and 1 U alkaline phosphatase (NEB). The reaction was kept at 37 °C for 3 h, and then the solution of pre-treated nucleosides was de-proteinized using a Sartorius 10 000-Da MWCO spin filter. Analysis of the nucleoside mixtures was performed using an Agilent 6460 QQQ mass spectrometer with an Agilent 1260 HPLC system. The multi-reaction monitoring (MRM) mode was used because of the high selectivity and sensitivity attained when working with parent-to-product ion transitions. The LC-MS data were acquired using the Agilent Qualitative Analysis software. The MRM peaks of each modified nucleoside were extracted and normalized to quantify the tRNA modifications (Yan et al., 2013; Su et al., 2014). Three technical replicates were used.