核糖核酸
计算生物学
亚细胞定位
生物
细胞分离
转录组
深度测序
细胞生物学
细胞质
基因
基因表达
生物化学
基因组
酶
作者
Fabienne Lefebvre,Neal Cody,Louis Philip Benoit Bouvrette,Julie Bergalet,Xiaofeng Wang,Éric Lécuyer
出处
期刊:Methods
[Elsevier]
日期:2017-08-01
卷期号:126: 138-148
被引量:23
标识
DOI:10.1016/j.ymeth.2017.05.017
摘要
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.
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