Isolation and Purification of tRNA

Isolation and purification of tRNA from C. reinhardtii and S. cerevisiae was performed as previously described (Alings et al. 2015 (link)). C. reinhardtii tRNA was additionally subjected to gel extraction from a denaturing 8 M urea 8% polyacrylamide gel (Lecanda et al. 2016 (link)). Subsequently, tRNA was enzymatically digested and dephosphorylated to yield monoribonucleosides (Alings et al. 2015 (link)). Briefly, 10 µg of tRNA was combined with 40 mU of Nuclease P1 from Penicillium citrinum (Sigma-Aldrich Biochemie GmbH) resuspended in 0.2 M CH₃CO₂Na (sodium acetate) pH 5.3 and 0.1 U of Shrimp Alkaline Phosphatase (Thermo Scientific) in 30 µL reactions at 37°C containing 2 mg/mL ZnCl₂ and 1×NEB3 buffer (New England Biolabs GmbH) for near neutral reaction conditions, or 20 mM CH₃CO₂Na pH 5.3 for acidic conditions. After 1.5 h the reaction mixture was supplemented with 15 µL 0.5 M NH₄HCO₃ and incubation at 37°C was resumed for 1 h. The reaction was terminated by adding 5.0% C₂HF₃O₂ (trifluoroacetic acid; TFA; Sigma-Aldrich Chemie GmbH) in water to a final concentration of 1.0%. The ribonucleosides were purified with HyperSep Hypercarb SPE Spin Tips (Thermo Fisher Scientific GmbH), dried to completion in a Savant SpeedVac concentrator (Thermo Fisher Scientific GmbH), and finally resuspended in 5 mM NH₄HCO₂ pH 5.3.