Division of Infectious Diseases, Department of Pediatrics, Center for Antimicrobial Resistance and Microbial Genomics, McGovern Medical School, University of Texas Health Sciences Center at Houston, Houston, TX, USA.
Methods Mol Biol. 2020;2136:113-133. doi: 10.1007/978-1-0716-0467-0_8.
Global gene expression analyses in bacteria have undergone a dramatic transformation. Prior to the development of high-throughput sequencing technologies, real-time PCR or microarray studies were the mainstay of assessing differences in gene expression in bacteria. Real-time PCR remains a critical tool for targeted gene expression analyses. However, microarray studies have given way to the plethora of advantages in RNA sequencing (RNA-seq) for the determination of global gene expression (i.e., transcriptome). Increased accessibility to high-throughput sequencing and user-friendly bioinformatics data analysis software have made RNA-seq technology use more widespread. Here, we provide comprehensive methods to perform RNA sequencing of Streptococcus pyogenes strains grown in vitro in standard laboratory media, including cell growth, RNA extraction, ribosomal RNA depletion, and library construction. Considerations for library sequencing and data analysis are also provided.
细菌的全球基因表达分析经历了巨大的转变。在高通量测序技术发展之前，实时 PCR 或微阵列研究是评估细菌中基因表达差异的主要方法。实时 PCR 仍然是靶向基因表达分析的关键工具。然而，微阵列研究已经让位于 RNA 测序（RNA-seq）在确定全局基因表达（即转录组）方面的众多优势。高通量测序的可及性增加以及用户友好的生物信息学数据分析软件使 RNA-seq 技术的应用更加广泛。在这里，我们提供了全面的方法来对在标准实验室培养基中体外生长的酿脓链球菌菌株进行 RNA 测序，包括细胞生长、RNA 提取、核糖体 RNA 耗尽和文库构建。还提供了文库测序和数据分析的注意事项。
