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  • 1
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2018, Vol.1737, pp.59-75
    Description: Transcriptomics, i.e., the quantification of cellular RNA transcripts, is a powerful way to gauge the physiological state of either bacterial or eukaryotic cells under a given condition. However, traditional approaches were unsuitable to measure the abundance of transcripts across kingdoms, which is relevant for biological processes such as bacterial infections of mammalian host cells. This changed with the establishment of "Dual RNA-seq," which profiles gene expression simultaneously in an infecting bacterium and its infected host. Here, we describe a detailed Dual RNA-seq protocol optimized for-but not restricted to-the study of human cell culture models infected with the Gram-negative model pathogen Salmonella Typhimurium. Furthermore, we provide experimental data demonstrating the benefits of some of the key steps of this protocol, including transcriptome stabilization (RNA fixation), FACS-based enrichment of invaded cells, and double rRNA depletion. While our focus is on data generation, we also include a section describing suitable computational methods to analyze the obtained datasets.
    Keywords: Cell Sorting ; Dual RNA-Seq ; Fixation ; Host-Pathogen Interaction ; Infection ; Noncoding RNA ; RNA-Seq ; Salmonella ; Transcriptomics ; Rrna Depletion ; Gene Expression Profiling ; Host-Pathogen Interactions ; Computational Biology -- Methods ; High-Throughput Nucleotide Sequencing -- Methods ; Salmonella Infections -- Microbiology ; Salmonella Typhimurium -- Genetics ; Sequence Analysis, RNA -- Methods
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 2
    Language: English
    In: Molecular Cell, 07 June 2018, Vol.70(5), pp.785-799
    Description: Bacteria are an exceedingly diverse group of organisms whose molecular exploration is experiencing a renaissance. While the classical view of bacterial gene expression was relatively simple, the emerging view is more complex, encompassing extensive post-transcriptional control involving riboswitches, RNA thermometers, and regulatory small RNAs (sRNAs) associated with the RNA-binding proteins CsrA, Hfq, and ProQ, as well as CRISPR/Cas systems that are programmed by RNAs. Moreover, increasing interest in members of the human microbiota and environmental microbial communities has highlighted the importance of understudied bacterial species with largely unknown transcriptome structures and RNA-based control mechanisms. Collectively, this creates a need for global RNA biology approaches that can rapidly and comprehensively analyze the RNA composition of a bacterium of interest. We review such approaches with a focus on RNA-seq as a versatile tool to investigate the different layers of gene expression in which RNA is made, processed, regulated, modified, translated, and turned over. RNA-seq-based approaches are revolutionizing how bacterial RNA biology can be studied. Hör, Gorski, and Vogel review the available global methods that can be used to chart the increasingly diverse number of RNA species and functions in any microbe of interest.
    Keywords: RNA-Seq ; Non-Coding RNA ; Small RNA ; Transcription ; RNA-Binding Protein ; Post-Transcriptional Control ; Biology
    ISSN: 1097-2765
    E-ISSN: 1097-4164
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  • 3
    Language: English
    Description: To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or non-coding RNAs are harder to detect. RNA-Seq has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-Seq data in order to generate high-resolution annotations is challenging, time consuming and requires numerous different steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-Seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-Seq and dRNA-Seq, predicts and annotates numerous features, including small non-coding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/....
    Keywords: Software ; Transcriptomic ; Genome Annotation ; Rna-Seq ; Transcriptomics
    Source: DataCite
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