摘要
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.
摘要译文
转录组学，即细胞RNA转录物的定量，是在给定条件下测量细菌或真核细胞的生理状态的有效方式。然而，传统方法不适合测量跨越王国的转录本的丰度，这与生物过程如哺乳动物宿主细胞的细菌感染有关。这随着“双RNA-seq”的建立而改变，其在感染细菌及其感染的宿主中同时描述基因表达。这里，我们描述了详细的双RNA-seq方案，该方案针对 - 但不限于 - 用革兰氏阴性模型病原体鼠伤寒沙门氏菌感染的人细胞培养模型的研究进行了优化。此外，我们提供实验数据，证明该协议的一些关键步骤的好处，包括转录组稳定化（RNA固定），基于FACS的入侵细胞富集和双rRNA消耗。我们的重点是数据生成，我们还包括一个描述合适的计算方法的部分来分析获得的数据集。
Alexander J. Westermann[1];Jörg Vogel[1]. Host-Pathogen Transcriptomics by Dual RNA-Seq. Bacterial Regulatory RNA[M].DE: Springer, 2018: 59-75