Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec, Canada; Département de Biochimie, Université de Montréal, Montréal, Québec, Canada.
Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec, Canada.
Methods. 2017 Aug 15;126:138-148. doi: 10.1016/j.ymeth.2017.05.017. Epub 2017 Jun 2.
The subcellular trafficking of RNA molecules is a conserved feature of eukaryotic cells and plays key functions in diverse processes implicating polarised cellular activities. Large-scale imaging and subcellular transcriptomic studies suggest that regulated RNA localization is a highly prevalent process that appears to be disrupted in several neuromuscular disorders. These features underline the importance and usefulness of implementing procedures to assess global transcriptome subcellular distribution properties. Here, we present a method combining biochemical fractionation of cells and high-throughput RNA sequencing (CeFra-seq) that enables rapid and efficient systematic mapping of RNA cytotopic distributions in cells. The described procedure involves biochemical fractionation to derive extracts of nuclear, cytosolic, endomembrane, cytoplasmic insoluble and extracellular material from cell culture lines. The RNA content of each fraction can then be profiled by deep-sequencing, revealing global subcellular signatures. We provide a detailed protocol for the CeFra-seq procedure along with relevant validation steps and data analysis guidelines to graphically represent RNA spatial distribution features. As a complement to imaging approaches, CeFra-seq represents a powerful and scalable tool to investigate global alterations in RNA trafficking.
RNA 分子的亚细胞转运是真核细胞的一个保守特征，在涉及极化细胞活动的多种过程中发挥关键作用。大规模成像和亚细胞转录组学研究表明，受调控的 RNA 定位是一种高度普遍的过程，似乎在几种神经肌肉疾病中被破坏。这些特征强调了实施程序以评估全局转录组亚细胞分布特性的重要性和有用性。在这里，我们提出了一种结合细胞生化分离和高通量 RNA 测序（CeFra-seq）的方法，该方法能够快速有效地系统绘制细胞中 RNA 细胞定位分布。所描述的程序涉及生化分离，从细胞培养系中提取核、胞质、内膜、细胞质不可溶和细胞外材料的提取物。然后可以通过深度测序对每个馏分的 RNA 含量进行分析，从而揭示全局亚细胞特征。我们提供了 CeFra-seq 程序的详细方案以及相关验证步骤和数据分析指南，以图形方式表示 RNA 空间分布特征。作为对成像方法的补充，CeFra-seq 是一种强大且可扩展的工具，可用于研究 RNA 运输的全局变化。
