Project description:We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain RNA, and liver FFPE RNA, and demonstrated that chemical capping in conjunction with template switching consistently reduces sequencing bias and improves library quality in comparison with commercially available RNA-seq kits. Finally, we showed the simultaneous detection of miRNAs and mRNAs in FFPE derived samples, underscoring the potential of this workflow for dissecting regulatory networks between miRNAs and their target gene transcripts.
Project description:MiRNAs are non-coding RNAs that regulate gene expression. MiRNAs mostly localise within the cytosol but are also found in the mitochondria where they can regulate the expression of mitochondrial-encoded transcripts. Small RNA library preparation protocols are well described when using total cellular RNA as the template, however, these methods are not directly applicable to total RNA extracted from fractionated cells, such as isolated mitochondria. The aim of this study was to optimise the small RNA library preparation protocol for use with small (<60ng) amounts of total mitochondrial RNA.
Project description:DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.
2014-01-28 | GSE46260 | GEO
Project description:Comparison of RNASeq Library Preparation Kits on FFPE Samples
Project description:DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability. BrdU and proteins ChIP-chip analyses analysis were carried out as described (Bermejo et al., 2009). Labelled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.
Project description:RNAseq experiments of Enterovirus A71 wild type demonstrate that the pyrazine-carboxamide ribonucleotide stimulates catalyzed intra- and intermolecular template switching. These results suggest that pyrazine-2 carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination, but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
Project description:Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice receiving non-toxic and toxic LNA gapmers after a single and repeat administration.
Project description:Deep Sequencing of mRNA from Drosophila melanogaster, Drosophila Sechellia, and their F1 hybrid. These data were generated in a study to analyze the extent and specificity of trans-splicing in Drosophila. mRNA-seq libraies were prepared from poly(A)+ RNA prepared from whole F1 hybrids of D. melanogaster and D. sechellia (female 0-3d post eclosion). Separate control libraries were prepared from D. melanogaster and D. sechellia total RNA (mixed before library preparation). The results of this study identified 80 novel trans-splicing events between homologous alleles of the same gene. Keywords: RNA-Seq Keywords: Expression profiling by high throughput sequencing There were 2 total samples in this study. Paired-end mRNA-seq was used to survey trans-splicing in F1 hybrids of D. melanogaster and D. sechellia. A mixed "control" library was used to assess the frequency of false-positive trans-splicing signal resulting from errors in reference genomes, high-throughput sequencing, and RT-PCR amplification based strand-switching.