Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany.
Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt, Germany.
Methods. 2020 Jun 1;178:49-62. doi: 10.1016/j.ymeth.2019.11.008. Epub 2019 Nov 18.
Precise knowledge on the binding sites of an RNA-binding protein (RBP) is key to understanding the complex post-transcriptional regulation of gene expression. This information can be obtained from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments. Here, we present a complete data analysis workflow to reliably detect RBP binding sites from iCLIP data. The workflow covers all steps from the initial quality control of the sequencing reads up to peak calling and quantification of RBP binding. For each tool, we explain the specific requirements for iCLIP data analysis and suggest optimised parameter settings.
精确了解 RNA 结合蛋白 (RBP) 的结合位点对于理解基因表达的复杂转录后调控至关重要。可以通过单核苷酸分辨率的紫外线交联和免疫沉淀 (iCLIP) 实验获得这些信息。在这里，我们提出了一个完整的数据分析工作流程，可从 iCLIP 数据中可靠地检测 RBP 结合位点。该工作流程涵盖了从测序读取的初始质量控制到峰调用和 RBP 结合的定量的所有步骤。对于每个工具，我们都解释了 iCLIP 数据分析的特定要求，并建议了优化的参数设置。
