library_construction_protocol: Total RNA was used to construct libraries for tRNA sequencing as previously described for multiplex small RNA-seq (MSR-seq) (PMID: 35513407). tRNA libraries: Briefly, total RNA was deacylated with a modified beta-elimination protocol  wherein  sodium periodate was omitted, but  high pH sodium tetra borate  was used to deacylated tRNA. tRNAs were ligated to a biotinylated hairpin oligo with a barcode and Illumina sequencing primer binding sites overnight. Reaction conditions were overnight at 16° C in a 50 μl ligation solution with the following final concentration: 15% PEG 8000, 1× NEB T4 RNA ligase I buffer, 50 μM ATP, 5% DMSO, 1 mM hexaamine cobalt (III) chloride, 0.8 μM barcode ligation oligo, and 1 U/μl NEB T4 RNA ligase I. After ligation, 50 μl of 100 mM EDTA were added to each sample to inactivate the ligase. The samples were then combined and 8 μl of ThermoFisher Streptavidin MyOne C1 Dynabeads per sample. The biotinylated samples were allowed to bind the beads for 15 min. After binding, the beads were magnetized, the supernatant was removed, and the samples were washed once with a high salt Tween wash buffer (1 M NaCl, 0.1% Tween 20, and 20 mM Tris–HCl, pH 7.4) and once with low salt wash buffer (100 mM NaCl, 20 mM Tris–HCl, pH 7.4). At this point, beads from  multiple samples were combine into a single tube. After combining, the sample was divided in two; one portion was demethylated with AlkB the other was untreated. For AlkB treatment, three stock solutions are made fresh immediately before reaction: L-ascorbic acid 200 mM, 2-ketoglutarate 3 mM, and ammonium iron sulfate 5 mM. The final reaction mixture contained 2 mM L-ascorbic acid, 1 mM 2-ketoglutarate, 0.3 mM ammonium iron sulfate, 100 mM KCl, 50 mM MES pH 6, 50 ng/μL BSA, 4 μM wild-type AlkB, and 4 μM AlkB-D135S. About 50 μL of the reaction mixture was added to 5 –20 μL of decanted streptavidin bead slurry after ligation, immobilization, and washing. The reaction continued for 30 min at 37 °C. Following the reaction, beads were washed once with high salt wash buffer (1 M NaCl, 20 mM Tris-HCl pH 7.4) and once with low salt wash buffer (100 mM NaCl, 20 mM Tris-HCl pH 7.4). Following AlkB treatment, both the treated and untreated samples were used for reverse transcription. The RT reaction was done with Superscript IV at 55° C for 10 min, then further incubated at 37 °C overnight. Beads were then resuspended into 50 μl of RNase H master mix containing 0.4 U/μl RNaseH (NEB) and 1× NEB RNase H buffer and incubated at 37° Cfor15 min, then washed. Unextended hairpin-oligo was blocked by addition of 10 μl of 250 mM freshly prepared sodium periodate, 0.5 M sodium acetate, pH 5 were added to the RNase H digested sample and incubated at room temperature for 30 min. Afterwards, ribose was added to a final concentration of 167 mM to quench excess periodate at room temperature for 5 min. Beads were resuspended into 50 μl of a ligation master mix with the following components: 2 U/μl T4 RNA ligase I (NEB), 1x NEB T4 RNA ligase I buffer, 2 μM second ligation oligo, 25% PEG 8000, 50 μM ATP, 7.5% DMSO and 1 mM hexaamine cobalt chloride. After incubation at room temperature overnight (> 12 h), the reaction was diluted with 50 μl water to reduce viscosity, washed once with high salt wash buffer and once with low salt wash buffer, and then resuspended in water. These beads were used as template for PCR reaction to make libraries for sequencing.