RNA oxidation and β-elimination

To measure tRNA charging levels, RNA oxidation and β-elimination were performed as described (Evans et al., 2017) with minor modifications. 25 μg of total RNA were resuspended in 10 mM sodium acetate pH 4.5 and oxidized by the addition of freshly prepared NaIO₄ to a final concentration of 50 mM in a 58 μL volume for 30 min at 22°C. The reaction was quenched by addition of 6 μL 1 M glucose for 5 min at 22°C. RNA was purified with Micro Bio Spin P30 columns (BioRad) followed by two rounds of ethanol precipitation in the presence of 0.3M sodium acetate pH = 4.5. Pellets were resuspended in 20 μL RNase-free water and β-elimination was performed by addition of 30 μl 100 mM sodium borate pH = 9.5 (freshly prepared) for 90 min at 45°C. RNA was recovered with Micro Bio Spin P30 columns followed by ethanol precipitation, resuspended in RNase-free water, quantified on a NanoDrop, and stored at −80°C in single-use aliquots.