Project description:We analyze whether tRNA quantifications based on small RNA-seq data are informative enough to distinguish between different human cell lines covering multiple tissue types. We therefore apply both small RNA-seq and Hydro-tRNAseq to HEK293 (kidney), HCT116 (colon), HeLa (cervix), MDA-MB-231 (breast), and BJ fibroblasts. First, the correlations between the two methods of identical samples and computational mapping pipeline range between 0.93 and 0.96 for all cell lines. tRNA quantifications from both protocols are compared and significantly higher Spearman correlations are obtained within matching samples versus mismatching cell lines. In consequence, we demonstrate that small RNA-seq quantifications of sample-specific tRNA profiles show a good agreement with conventional tRNA-seq.
Project description:We measured abundances of tRNAs by means of hydro-tRNA-seq (Gogakos et al., 2017), a method based on partial alkaline RNA hydrolysis that generates fragments suitable for sequencing, in the genome-reduced bacterium Mycoplasma pneumoniae.
Project description:To investigate differential mitochondrial tRNA gene expression in wild type (WT) and mutant (E423P) mitochondrial RNA polymerase (POLRMT) overexpression flies, we performed Drsophila mitochondrial tRNA-seq according to the Hydro-tRNAseq method. tRNA-seq reads were subjected to 3' - adapter filtering , 3' - adapter trimming and quality control. The tRNAs expression levels were measured by tag count. For each tRNA sequence-based profile, the mapped reads number used to estimate the expression level of each tRNA. The tRNAs expression profiling was calculated based on uniquely mapped reads and including mapped reads, respectively. We found no difference in the relative abundance of mitochondrial individual tRNAs between WT and E423P overexpression flies.
Project description:To associate the amount of tRNAs with codon usage, we perform hydro-tRNA sequencing (Gogakos et al. 2017) and quantify tRNA expression in HEK293 and HeLa cells.
Project description:To quantify tRNA expression, we perform hydro-tRNA sequencing (Gogakos et al. 2017) in HACAT (keratinoytes) and HepG2 (liver) human cell lines. This study contains 6 samples. Three replicates for each of the five cell lines. This data complements our previously published data (GEO: GSE137834), which contained five additional cell lines from different tissues: HEK293 (kidney), HCT116 (colon), HeLa (cervix), MDA-MB-231 (breast), and BJ fibroblasts. Therefore, with these extra two cell lines, this constitutes a tissue-wide dataset of tRNA sequencing covering a total of seven human cell lines.
Project description:We report the identification and quantification of Watson-Crick modifications in tRNA and rRNA through the use of high throughput sequencing. We apply the recently published DM-tRNA-Seq method to generate demethylase treated and untreated 293T samples, and using computational methods we are able to flag sites using a modification index. This index allows us to generate site-resolved information about modification that we can use to identify and quantify Watson-Crick face modifications in tRNA and rRNA. With the demethylase treated samples, we are able to validate numerous nucleotide modifications from demethylase substrates, and the absence of demethylase action also serves to aid in identification. We find numerous novel modification sites in tRNA as well as striking comparisons between tissues cultures lines. Our study reports a comprehensive analysis of the tRNA modification landscape by identifying sites of modification as well as quantifying modification levels.
Project description:High-resolution quantitative profiling of tRNA abundance and modification status in hematopoietic cells upon Trmt6 deletion by mim-tRNAseq
Project description:The participation of transfer RNAs (tRNAs) in fundamental aspects of biology and disease necessitates an accurate, experimentally confirmed annotation of tRNA genes, and curation of precursor and mature tRNA sequences. This has been challenging, mainly because RNA secondary structure and nucleotide modifications, together with tRNA gene multiplicity, complicate sequencing and sequencing read mapping efforts. To address these issues, we developed hydro-tRNAseq, a method based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing. To identify transcribed tRNA genes, we further complemented this approach with Photoactivatable Crosslinking and Immunoprecipitation (PAR-CLIP) of SSB/La, a conserved protein involved in pre-tRNA processing. Our results show that approximately half of all predicted tRNA genes are transcribed in human cells. We also report predominant nucleotide modification sites, their order of introduction, and identify tRNA leader, trailer and intron sequences. By using complementary sequencing-based methodologies, we present a human tRNA atlas, and determine expression levels of mature and processing intermediates of tRNAs in human cells.
Project description:Measurements of cellular tRNA abundance are hampered by pervasive blocks to cDNA synthesis at modified nucleosides and the extensive similarity among tRNA genes. We overcome these limitations with modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which combines a workflow for full-length cDNA library construction from mature tRNA with a simple-to-use computational analysis toolkit. Our method accurately captures tRNA abundance and modification status in multiple eukaryotes and is applicable to any organism with a known genome.