tRNA and tRNA fragment (tRF) sequencing

Total tRNA, ribosome-contained tRNA, and tRF sequencing analyses were performed essentially as described (20). For total tRNA and tRF preparation, total RNA was first prepared from brain lysate (also used for ribosome profiling and transcriptome analysis) using TRI Reagent. Total RNA was resolved by denaturing urea PAGE, and tRNA (60 to 100 nt) and tRF (20 to 40 nt) were collected by gel excision. To collect ribosome-contained tRNA, during gel excision of mRNA fragments (20 to 40 nt) in ribosome profiling, tRNA fragments (60 to 100 nt) were also collected. Total tRNA, ribosome-contained tRNA, and tRF were demethylated using Escherichia coli–derived Alpha-ketoglutarate-dependent dioxygenase (AlkB) to enable reverse transcription (56). Briefly, tRNA or tRF were incubated in 45 mM tris-HCl (pH 8), 0.9 mM α-ketoglutaric acid, 1.8 mM ascorbic acid, 67 μM (NH₄)₂Fe(SO₄)₂, and 2.5 μM AlkB, at 37°C for 2 hours. Demethylation was confirmed by performing yeast tRNA demethylation and RNA nucleoside mass spectrometry analysis, as a positive control. Subsequently, library preparation was performed in the same way as mRNA library preparation. After Illumina MiSeq sequencing, tRNA and tRF analysis was performed using Galaxy. Briefly, fastq files were converted to fasta files, 12 nt of adaptor sequence (GGCACCATCAAT) was clipped (leaving 5 nt of 3′ adaptor sequence CTGTA at the tRNA 3′ end to allow mapping of short reads), and tRNA reads were Bowtie2-aligned to mouse tRNA sequences in the Genomic tRNA database (57). Reads were counted using alignment position information in the SAM file and count function in Galaxy.