In vitro synthesis of tRNAs: The plasmid containing tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis sequence, respectively, was linearized with NsiI, and 3 µg of the linear plasmid were used for in vitro transcription by T7 RNA polymerase (Thermo Fisher Scientific) according to the manufacturer's instructions. After a 7 h incubation at 37°C and subsequent DNase I treatment, the transcription reaction was purified using phenol/ chloroform/ isoamylalcohol extraction followed by gel filtration on Sephadex G50 (GE Healthcare). Extraction of small RNAs from cells: Small RNAs were extracted according to the manufacturer's protocol (PureLink™ miRNA Isolation Kit (Invitrogen)) with some adjustments. Briefly, cells were suspended in 1 ml Trizol (Ambion). The samples were vortexed for 2 min after adding 0.2 ml chloroform and glass beads, and then were centrifuged at 16 000 rcf, 4°C for 15 min. After adding 215 µl ethanol to the upper phase, the samples were transferred to a spin cartridge and centrifuged at 12 000 rcf for 1 min. 700 µl of ethanol was added to the flow-through, and the samples were transferred to a new spin cartridge followed by centrifugation at 12 000 rcf for 1 min. After washing the cartridge with wash buffer, small RNAs were eluted with 50 µl of DEPC-treated water. For library preparation, 20 pmol of in vitro-transcribed tRNAAsp were used as input material. To promote ligation of sequencing adapters to tRNA, 16 pmol of custom double-stranded splint adapter (with CGGU overhang complimentary to 3' termini of tRNAAsp GCCA) were used for the first ligation. The first ligation reaction was performed in a DNA LoBind tube at 25°C for 45 min. It also contained 1 x RNA ligase2 buffer (New England Biolabs), 5% PEG 8000, 2 mM ATP, 6.25 mM MgCl2, 6.25 mM DTT, and 0.5 units/µl T4 RNA ligase 2 (10 000 units/ml) in a total volume of 20 µl. For library preparation of mixtures of in vitro-transcribed tRNAs, 10 pmol of each of the four tRNAs (tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis) and 16 pmol of each of the two splint adapters (splint adapters with CGGU and UGGU overhang) were used in a reaction volume of 40 µl. Library preparation for tRNAs isolated from cells was performed using 40 pmol of deacylated small RNAs and 32 pmol of splint adapter (8 pmol of each of the four splint adapters). Ligation reaction samples were subsequently electrophoresed on a 7 M urea/TBE PAGE gel (8%), and ligation products were excised and purified from the gel. The concentration of gel-purified ligation products was measured with the Qubit RNA HS Assay Kit (Invitrogen). The second ligation of splint-ligated tRNAs to the RNA Adapter (RMX, included in the SQK-RNA002 kit, Oxford Nanopore Technologies) and subsequent library purification were performed following the Nanopore Sequence-Specific Direct RNA sequencing protocol, except that incubation time of the second ligation was increased from 10 min to 30 min to improve ligation efficiency. Direct RNA nanopore sequencing