Enzymatic Hydrolysis and LCMS Analysis of tRNA

tRNA was digested using 0.6 U nuclease P1 (Sigma-Aldrich) in the presence of ZnCl₂, ammonium acetate pH 5.3, THU, pentostatine and BHT from²¹ (link) at 50°C for one hour followed by overnight incubation at room temperature. The next day, 10 U benzonase (Sigma-Aldrich, Munich, Germany), 0.1 U phosphodiesterase I (VWR, Ismaning, Germany) and MgCl₂ in a final concentration of 1 mM were added and incubated at 37°C for one hour. Then 20 U alkaline phosphatase (Sigma-Aldrich, Munich, Germany) and the proper amount to reach a final concentration of 0.1 M Tris-HCl pH 8 were added. After 2 hours at 37 °C the digestion mix was filtered through a 10 kDa MWCO filter (AcroPrep™ Advance, 350 µl, Omega™ 10K MWCO, Pall, Dreieich, Germany) at 3000 xg for 30 minutes. 18 µL of filtrate was mixed with 2 µL of the 10x SILIS and subjected to LC-MS/MS analysis.