CeFra-seq: Systematic mapping of RNA subcellular distribution properties through cell fractionation coupled to deep-sequencing

Methods. 2017 Aug 15;126:138-148. doi: 10.1016/j.ymeth.2017.05.017. Epub 2017 Jun 2.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Fractionation
  • Cell Nucleus / genetics*
  • Cell Nucleus / metabolism
  • Chromosome Mapping / methods*
  • Cytoplasm / genetics*
  • Cytoplasm / metabolism
  • Drosophila
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • K562 Cells
  • RNA / genetics*
  • RNA / isolation & purification
  • Sequence Analysis, RNA / methods*

Substances

  • RNA