library_construction_protocol: Cells were maintained in 60mm dishes at 37 °C in a humidified atmosphere at 5% CO2 in DMEM 4.5g/L Glucose with UltraGlutamine media supplemented with 10% of Tet-free FBS (Clontech) and 1% penicillin/streptomycin. Cells were washed twice with PBS and added 700µL of Trizol and continue with the extraction protocol. Total RNA from HEK293 and HeLa was extracted using the miRNeasy Mini kit. Total RNA was resolved on 15% Novex TBE-urea gels and size-selected for 60-100 nt fragments. The recovered material was then alkaline hydrolyzed (10 mM sodium carbonate and 10 mM sodium bicarbonate) for 10 min at 60°C. Treated following the protocol of hydro-tRNAseq (Gogakos et al. 2017) The resulting RNA was de-phosphorylated with Antarctic Phosphatase (New England Biolabs) at 37°C for 1 h. De-phosphorylated RNA was purified with an RNeasy MinElute spin column and re-phosphorylated with polynucleotide kinase (NEB). PNK-treated tRNAs were purified with an RNeasy MinElute spin column, adaptor-ligated, reverse-transcribed, and PCR-amplified for 14 cycles. The resulting cDNA was purified using a QIAQuick PCR Purification Kit.