tRNA-seq of DUS1L knockout and wild-type A375 human melanoma cells
tRNA-seq was performed on A375 human melanoma cells with CRISPR/Cas9-mediated knockout of DUS1L and wild-type control cells, with three biological replicates per group. Total RNA was extracted using TRIzol, and ~20 µg RNA per sample was fractionated on 12% urea-PAGE to isolate 60–100 nt RNA fragments. These fragments were subjected to demethylation of m1A, m1G and m3C using an RNA Pretreatment Kit, followed by partial alkaline hydrolysis, dephosphorylation and re-phosphorylation. Small RNA libraries were constructed from 19–35 nt fragments using the NEBNext Small RNA Library Prep Set for Illumina and sequenced on an Illumina NextSeq 500 platform. Clean reads were aligned with BWA to human mature and genomic tRNA reference sequences compiled from GtRNAdb, and uniquely mapped reads were summarized to generate tRNA expression profiles for differential expression analysis between DUS1L knockout and wild-type cells. Overall design: Six tRNA-seq libraries were generated from A375 cells: three biological replicates of wild-type cells and three biological replicates of DUS1L knockout cells. Size-selected small RNAs were used to construct NEBNext-based small RNA libraries, which were sequenced on an Illumina NextSeq 500 platform. Differential expression analysis of tRNA species between DUS1L knockout and wild-type cells was performed using DESeq2.