Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP)
Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP)
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DOI：
10.1007/978-1-4939-7204-3_14
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发表时间：
2017-01-01
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影响因子：
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通讯作者：
Yeo, Gene W.
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文献类型：
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作者：
Van Nostrand, Eric L.;Nguyen, Thai B.;Yeo, Gene W.
体内 RNA 结合蛋白靶标的分析为了解它们在调节 RNA 加工中发挥的机制作用提供了重要的见解。增强交联和免疫沉淀 (eCLIP) 方法为转录组范围内的蛋白质-RNA 相互作用的稳健、可重复的鉴定提供了一个框架，与以前的方法相比，效率显着提高。在这里，我们提供了 eCLIP 方法的分步描述，以及对协议中关键步骤最佳性能的见解。特别是，我们描述了对适配器策略的改进，该策略可实现单端增强 CLIP (seCLIP)，从而消除了对 eCLIP 库双端测序的要求。此外，我们描述了对标准硝酸纤维素膜供应商中存在的污染 RNA 的观察，并为敏感应用提供了显着减少污染的选项。这些说明进一步完善了 eCLIP 方法，为所有用户简化了可靠的 RNA 结合蛋白研究。
Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.