Identification of m6A residues at single nucleotide resolution using eCLIP and an accessible custom analysis pipeline

We have developed a modified eCLIP-based method (meCLIP) to identify m6A residues at single-nucleotide resolution. By coupling the improvements of eCLIP with an easy-to-use computational pipeline, we have successfully identified over 50,000 unique m6A residues in the two breast cancer cell lines that were analyzed (MCF-7 and MDA-MB-231) and over 8,000 unique residues in HEK-293 cells. We compared these residues to the sites called using the currently most utilized m6A identification method (miCLIP). m6A identification in 3 cell lines (MCF-7, MDA-MB-231, and HEK-293) using modified eCLIP-based library preparation and custom analysis pipeline

我们研发了一种基于增强交联免疫沉淀（enhanced Cross-Linking Immunoprecipitation, eCLIP）的改良方法（meCLIP），用于在单核苷酸分辨率下鉴定N6-甲基腺苷（m6A）残基。通过将eCLIP的技术改良与易用的计算分析流程相结合，我们在所分析的两种乳腺癌细胞系（MCF-7与MDA-MB-231）中成功鉴定出超过50000个独特的m6A残基，在HEK-293细胞中则鉴定出超过8000个独特的m6A残基。我们将这些鉴定得到的残基与当前应用最为广泛的m6A鉴定方法miCLIP所获得的位点进行了比对分析。本研究采用基于改良eCLIP的文库制备流程与定制化分析流程，在MCF-7、MDA-MB-231及HEK-293这3种细胞系中开展了m6A鉴定工作。