Abstract
Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) allows the identification of RNA targets bound by a specific RNA-binding protein (RBP) in in vivo and ex vivo experimental models with high specificity. Due to the little RNA yield obtained after cross-linking, immunoprecipitation, polyacrylamide gel electrophoresis, membrane transfer, and RNA extraction, CLIP-seq is usually performed from relatively large amounts of starting material, like cell lysates or tissue homogenates. However, RBP binding of its specific RNA targets depends on its subcellular localization, and a different set of RNAs may be bound by the same RBP within distinct subcellular sites. To uncover these RNA subsets, preparation of CLIP-seq libraries from specific subcellular compartments and comparison to CLIP-seq datasets from total lysates is necessary, yet there are currently no available protocols for this. Here we describe the adaptation of CLIP-seq to identify the specific RNA targets of an RBP (FUS) at a small subcompartment, that is, neuronal synapses, including subcompartment isolation, RBP–RNA complex enrichment, and upscaling steps.
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Acknowledgments
We gratefully acknowledge the support from the animal facility of the University Hospital of Zurich (USZ) and the Short Read Sequencing group from the Genomics/Transcriptomics team at the Functional Genomics Center Zurich (FGCZ) from the University of Zurich (UZH). We thank Julien Weber for experimental support and the National Center of Competence in Research (NCCR), RNA & Disease funded by the Swiss National Science Foundation (SNF) for financial support.
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Sahadevan, S., Pérez-Berlanga, M., Polymenidou, M. (2022). Identification of RNA–RBP Interactions in Subcellular Compartments by CLIP-Seq. In: Matějů, D., Chao, J.A. (eds) The Integrated Stress Response. Methods in Molecular Biology, vol 2428. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1975-9_19
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DOI: https://doi.org/10.1007/978-1-0716-1975-9_19
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