title: GSM5664709: KO1_AspGTC; Mus musculus; OTHER
library_construction_protocol: 1ug of total RNA was used for bisulfite conversion with the EZ RNA Methylation kit according to manufacturer's instructions (Zymo Research). Due to the strong secondary structure of tRNAs, the PCR step during bisulfite conversion was repeated 3 times consecutively, as previously reported to completely denature tRNAs.  Following bisulfite treatment and clean-up, we performed cDNA synthesis using the reverse primer for each tRNA isodecoder.  Targeted cDNA was then PCR-amplified with the tRNA-specific forward primer and products were size-verified on a 4% agarose gel. PCR amplicons (~120nt including tRNA amplicon and overhangs for adapter ligation) were then extracted and purified (Qiaquick Gel Extraction Kit, Qiagen).  We labelled each amplicon with a unique index (Nextera XT, Illumina) and pooled up to 20 amplicons in equimolar concentrations before next-gen sequencing. Note that only read 1 from paired end sequencing was used for analysis. total RNA was extracted from mouse cortex using Trizol extraction and quantity was measured on Qubit fluorometer.  Quality of total RNA was checked on the Agilent Bioanalyzer using the RNA 6000 Pico Kit