RNA-binding proteins are important players in post-transcriptional regulation, such as modulating mRNA splicing, translation, and degradation under diverse biological settings. Identifying and characterizing the RNA substrates is a critical step in deciphering the function and molecular mechanisms of the target RNA-binding proteins. High-throughput sequencing of the RNA fragments isolated by crosslinking immunoprecipitation (CLIP-seq) is one of the standard techniques to identify the in vivo transcriptome-wide binding sites of the target RNA-binding protein. This method is widely used in functional and mechanistic characterizations of RNA-binding proteins. In this review, we provide several practical considerations on performing and analyzing CLIP-seq experiments. Particularly, we focus on how to perform CLIP-seq experiments on endogenous RNA-binding proteins. In addition, we provide a practical summary on how to choose and use computational pipelines from an increasing number of computational methods and packages that are available for analyzing the sequencing datasets from the CLIP-seq experiments. We hope these practical considerations will facilitate experimental biologists in performing and analyzing CLIP-seq experiment to obtain biologically relevant mechanistic insights.

中文翻译：

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执行和分析 CLIP-seq 实验以确定转录组范围内的 RNA-蛋白质相互作用的实际考虑因素


RNA 结合蛋白在转录后调节中发挥着重要作用，例如在不同的生物环境下调节 mRNA 剪接、翻译和降解。鉴定和表征 RNA 底物是破译目标 RNA 结合蛋白的功能和分子机制的关键步骤。通过交联免疫沉淀 (CLIP-seq) 分离的 RNA 片段的高通量测序是鉴定靶 RNA 结合蛋白的体内转录组范围结合位点的标准技术之一。该方法广泛用于 RNA 结合蛋白的功能和机制表征。在这篇综述中，我们提供了执行和分析 CLIP-seq 实验的一些实际考虑因素。我们特别关注如何在内源性 RNA 结合蛋白上进行 CLIP-seq 实验。此外，我们还提供了关于如何从越来越多的计算方法和包中选择和使用计算管道的实用总结，这些方法和包可用于分析 CLIP-seq 实验的测序数据集。我们希望这些实际考虑因素将有助于实验生物学家执行和分析 CLIP-seq 实验，以获得生物学相关的机制见解。