Westermann Alexander J, Vogel Jörg
Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
Helmholtz Institute for RNA-Based Infection Research (HIRI), Würzburg, Germany.
Methods Mol Biol. 2018;1737:59-75. doi: 10.1007/978-1-4939-7634-8_4.
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.
转录组学，即对细胞RNA转录本进行定量分析，是一种在给定条件下衡量细菌或真核细胞生理状态的有力方法。然而，传统方法不适用于跨物种测量转录本丰度，而这对于诸如哺乳动物宿主细胞的细菌感染等生物学过程至关重要。随着“双RNA测序”（Dual RNA-seq）技术的建立，这种情况得到了改变，该技术可同时对感染细菌及其被感染宿主中的基因表达进行分析。在此，我们描述了一种详细的双RNA测序方案，该方案针对感染革兰氏阴性模式病原体鼠伤寒沙门氏菌的人类细胞培养模型研究进行了优化，但并不局限于此。此外，我们提供了实验数据，证明了该方案一些关键步骤的益处，包括转录组稳定（RNA固定）、基于荧光激活细胞分选（FACS）对侵入细胞的富集以及双重核糖体RNA（rRNA）去除。虽然我们的重点是数据生成，但我们还包括一个章节，描述了用于分析所得数据集的合适计算方法。
