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优化PAR-CLIP以在全转录组范围内鉴定RNA结合蛋白的结合位点。

Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins.

作者信息

Garzia Aitor, Meyer Cindy, Morozov Pavel, Sajek Marcin, Tuschl Thomas

机构信息

Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.

Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.

出版信息

Methods. 2017 Apr 15;118-119:24-40. doi: 10.1016/j.ymeth.2016.10.007. Epub 2016 Oct 17.

DOI:10.1016/j.ymeth.2016.10.007
PMID:27765618
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5393971/
Abstract

Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in combination with next-generation sequencing is a powerful method for identifying endogenous targets of RNA-binding proteins (RBPs). Depending on the characteristics of each RBP, key steps in the PAR-CLIP procedure must be optimized. Here we present a comprehensive step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. Furthermore, we report the application of a new PAR-CLIP data analysis pipeline to three distinct RBPs targeting different annotation categories of cellular RNAs.

摘要

光激活核糖核苷增强交联与免疫沉淀技术（PAR-CLIP）结合新一代测序技术是一种用于鉴定RNA结合蛋白（RBP）内源性靶点的强大方法。根据每个RBP的特性，必须优化PAR-CLIP程序中的关键步骤。在此，我们提供了一份全面的PAR-CLIP分步方案，并对关键步骤进行了详细解释。此外，我们报告了一种新的PAR-CLIP数据分析流程在针对细胞RNA不同注释类别的三种不同RBP上的应用。

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