Comprehensive tRNA Profiling Protocol

tRNA profiling was conducted as previously described^{8 (link)}. Briefly, samples were deacylated with 0.1 M Tris-HCl (pH 9) at 37°C for 30 minutes, precipitated, and biotinylated using Pierce RNA 3’ End Biotinylation Kit (Thermo Fisher), with 0.66 pmol of yeast tRNA-Phe added as a spike-in standard. Following chloroform extraction, biotinylated RNA was hybridized to DNA probe pairs complementary to the 3’ and 5’ arms of each tRNA isoacceptor. Nicks at the anticodon loop of DNA-RNA hybrids were ligated using SplintR ligase and T4 DNA ligase (NEB). DNA-RNA hybrids were purified using My-One-C1 Streptavidin Dynabeads (Invitrogen), and ligated probes were eluted after Rnase H and Rnase A treatment. Probes were PCR amplified and sequenced using Illumina NextSeq (MidOutput, 150 SR) at the Rockefeller University Genomics Center. For computational analysis, fastq files were aligned to tRNA probe sequences using bowtie2, and reads were further sorted, indexed, and counts were generated with samtools. Raw counts were imported into R 4.0.2 and differentially expressed transcripts determined using DESeq2.