Van Nostrand Eric L, Gelboin-Burkhart Chelsea, Wang Ruth, Pratt Gabriel A, Blue Steven M, Yeo Gene W
Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Stem Cell Program, University of California at San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA.
Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Stem Cell Program, University of California at San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA; Bioinformatics and Systems Biology Graduate Program, University of California San Diego, La Jolla, CA, USA.
Methods. 2017 Apr 15;118-119:50-59. doi: 10.1016/j.ymeth.2016.12.007. Epub 2016 Dec 18.
Identification of in vivo direct RNA targets for RNA binding proteins (RBPs) provides critical insight into their regulatory activities and mechanisms. Recently, we described a methodology for enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP) using antibodies against endogenous RNA binding proteins. However, in many cases it is desirable to profile targets of an RNA binding protein for which an immunoprecipitation-grade antibody is lacking. Here we describe a scalable method for using CRISPR/Cas9-mediated homologous recombination to insert a peptide tag into the endogenous RNA binding protein locus. Further, we show that TAG-eCLIP performed using tag-specific antibodies can yield the same robust binding profiles after proper control normalization as eCLIP with antibodies against endogenous proteins. Finally, we note that antibodies against commonly used tags can immunoprecipitate significant amounts of antibody-specific RNA, emphasizing the need for paired controls alongside each experiment for normalization. TAG-eCLIP enables eCLIP profiling of new native proteins where no suitable antibody exists, expanding the RBP-RNA interaction landscape.
鉴定RNA结合蛋白(RBP)在体内的直接RNA靶标,能为了解其调控活性和机制提供关键见解。最近,我们描述了一种方法,即使用针对内源性RNA结合蛋白的抗体进行增强交联和免疫沉淀,随后进行高通量测序(eCLIP)。然而,在许多情况下,人们希望分析缺乏免疫沉淀级抗体的RNA结合蛋白的靶标。在此,我们描述了一种可扩展的方法,利用CRISPR/Cas9介导的同源重组将肽标签插入内源性RNA结合蛋白基因座。此外,我们表明,使用标签特异性抗体进行的TAG-eCLIP在经过适当的对照标准化后,能够产生与使用针对内源性蛋白的抗体进行的eCLIP相同的稳健结合图谱。最后,我们注意到,针对常用标签的抗体可以免疫沉淀大量抗体特异性RNA,这强调了每个实验都需要配对对照进行标准化。TAG-eCLIP能够在没有合适抗体的情况下对新的天然蛋白进行eCLIP分析,扩展了RBP-RNA相互作用的研究范围。