Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites [array]. Homo sapiens

RNA binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNAs. As defects in protein-RNA recognition lead to human disease, UV-crosslinking and immunoprecipitation (CLIP) of ribonuclear complexes followed by deep sequencing (-seq) is critical in constructing protein-RNA maps to expand our understanding of RBP function. However, current CLIP protocols are technically demanding and involve low complexity libraries that yield squandered sequencing of PCR duplicates and high experimental failure rates. To enable truly large-scale implementation of CLIP-seq, we have developed an enhanced CLIP methodology (eCLIP) that features a decrease of ~10 cycles of requisite amplification with a concomitant >60% decrease in discarded PCR duplicate reads, while maintaining the ability to identify RNA binding with single-nucleotide resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP also dramatically improves specificity in discovery of authentic binding sites. To demonstrate that eCLIP enables large-scale and robust profiling of RBPs, 102 eCLIP experiments in biological duplicate for a diverse collection of 74 RBPs in HepG2 and K562 cells were completed (available at https://www.encodeproject.org). We establish that eCLIP is comparable in amplification and sample requirements to ChIP-seq, and enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP experiments and RNA-centric perspectives of RBP activity. Overall design: Microarray profiling of RBFOX2 knockdown by shRNAs in 293T cells

RNA结合蛋白（RNA binding proteins, RBPs）通过与靶RNA相互作用，在细胞生理过程中发挥核心功能。由于蛋白质-RNA识别缺陷会引发人类疾病，因此对核糖核蛋白复合物进行紫外交联免疫沉淀（UV-crosslinking and immunoprecipitation, CLIP）后辅以深度测序（-seq），是构建蛋白质-RNA相互作用图谱、深化我们对RBP功能认知的关键技术。然而当前的CLIP实验方案技术门槛较高，且所得文库复杂度偏低，会造成PCR重复序列的测序资源浪费，同时实验失败率居高不下。为实现CLIP-seq的规模化应用，我们开发了一种增强型CLIP方法（enhanced CLIP methodology, eCLIP）：该方法将所需的扩增循环数减少约10次，同时丢弃的PCR重复序列读数占比降低60%以上，且仍可实现单核苷酸分辨率的RNA结合位点鉴定。通过简化配对IgG对照与尺寸匹配输入对照的制备流程，eCLIP还可显著提升鉴定真实结合位点的特异性。为验证eCLIP可实现RBPs的大规模、稳健的谱分析，我们在HepG2与K562细胞中针对74种多样化的RBP完成了102次生物学重复的eCLIP实验（相关数据可通过https://www.encodeproject.org获取）。我们证实，eCLIP在扩增需求与样本用量上与染色质免疫沉淀测序（ChIP-seq）相当，且可实现多种RBPs的整合分析，从而揭示因子特异性结合谱、CLIP实验常见的实验伪影，以及基于RNA视角的RBP活性特征。总体实验设计：利用短发夹RNA（short hairpin RNA, shRNAs）在293T细胞中敲低RBFOX2后的微阵列谱分析。