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

•CeFra-seq reveals transcriptome-wide subcellular and extracellular distribution properties through cell fractionation coupled to RNA-seq.•CeFra-seq results can be transposed using simple data transformation steps to yield simplex plots of transcriptome-wide distributions.•CeFra-seq provides a syste...

Full description

Saved in:
Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2017-08, Vol.126, p.138-148
Main Authors: Lefebvre, Fabio Alexis, Cody, Neal A.L., Bouvrette, Louis Philip Benoit, Bergalet, Julie, Wang, Xiaofeng, Lécuyer, Eric
Format: Article
Language:English
Subjects:
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
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:•CeFra-seq reveals transcriptome-wide subcellular and extracellular distribution properties through cell fractionation coupled to RNA-seq.•CeFra-seq results can be transposed using simple data transformation steps to yield simplex plots of transcriptome-wide distributions.•CeFra-seq provides a systematic and scalable alternative to imaging approaches in global analyses of RNA localization. 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.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2017.05.017