Isolation of mammalian stress granule cores for RNA-Seq analysis

• Published in: Methods (San Diego, Calif.) Vol. 137; pp. 49 - 54
• Main Authors: Khong, Anthony, Jain, Saumya, Matheny, Tyler, Wheeler, Joshua R., Parker, Roy
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.03.2018
• Subjects: Animals; In Situ Hybridization, Fluorescence - methods; Mammals - genetics; Purification; Ribonucleoproteins - genetics; RNA, Messenger - genetics; RNA-Seq; RNP granule; Sequence Analysis, RNA; Single molecule FISH; Single Molecule Imaging - methods; Stress granule; Stress, Physiological - genetics; Single molecule FISH; Stress granule; Purification; RNP granule; RNA-Seq
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2017.11.012

•SG cores can be biochemically purified by successive centrifugation and immunoprecipitation.•Validation of RNA-Seq results by single molecule FISH.•Image analysis for single molecule FISH using the Bitplane Imaris image analysis software. Stress granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian stress granules contain stable structures, referred to as “cores” that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from stress granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of stress granules and may assist in the discovery of the assembly mechanisms and functions of stress granules in a variety of biological contexts.