Total RNA, including small RNAs and tRNAs, was extracted from mouse brain tissue using TRIzol reagent. Tissues were homogenized in TRIzol followed by chloroform phase separation. The aqueous phase was precipitated overnight using sodium acetate (0.3M final concentration) and ethanol (70-75% final concentration) to maximize small-RNA recovery. The resulting RNA pellet was washed with 75% ethanol and resuspended in a 10 mM NaOAc, 1 mM EDTA (pH 4.8) buffer. Small RNA sequencing libraries were constructed by MesoRNA (Chicago, IL, USA) utilizing MSR-seq (PMID: 35513407) Multiplexed NGS library preparation workflow to simultaneously capture tRNA abundance, processing, aminoacylation, and modification signatures from a single assay.