Nuclease p1

tRNA^Phe from yeast was produced by in vitro transcription (33 (link)) and incubated with recombinant TruB (34 (link)) in MST 1× buffer (20 mM Tris pH 7.5, 60 mM KCl, 0.02% Tween-20 at 80°C for 70 min). For release of the RNA from the enzyme, the reaction mixture was boiled for 10 min at 95°C and subsequently dissolved in 20 mM NH₄OAc pH 5.3. The samples were incubated for 2 h at 70°C in the presence of 0.0003 U nuclease P1 (Roche Diagnostics, Mannheim, Germany) per 10 pmol RNA, which leads to a complete degradation to mononucleotides. Snake venom phosphodiesterase (Worthington, Lakewood, USA) was then added to a concentration of 0.06 U per 100 μg RNA, and the mixture was incubated at 37°C for another 1 h. Finally, to convert the resulting mixture of mononucleotides to free nucleosides, 1/10 volume of 10× FastAP buffer (Fermentas, St Leon-Roth, Germany) was added, followed by 3/20 volume of H₂O, and 1 U of FastAP Thermosensitive Alkaline Phosphatase (FastAP stock at 1 U/μl; from Fermentas, St Leon-Roth, Germany). The mixture was incubated for 1 h at 37°C and divided into two aliquots. One aliquot was spiked with 100 fmol pseudouridine, whereas the other was spiked with pure water, prior to adding 10 vol% of SIL-IS and subjecting the samples to LC-MS/MS analysis.