Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).
Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).
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DOI：
10.1038/nmeth.3810
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发表时间：
2016-06
期刊：
影响因子：
48
通讯作者：
Yeo GW
中科院分区：
文献类型：
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作者：
Van Nostrand EL;Pratt GA;Shishkin AA;Gelboin-Burkhart C;Fang MY;Sundararaman B;Blue SM;Nguyen TB;Surka C;Elkins K;Stanton R;Rigo F;Guttman M;Yeo GW
由于RNA结合蛋白（RBP）通过与靶RNA相互作用在细胞生理学中发挥重要作用，因此通过核糖核蛋白复合物的紫外交联和免疫沉淀（CLIP）识别结合位点对于了解RBP功能至关重要。然而，目前的CLIP协议在技术上要求很高，并且产生具有高实验失败率的低复杂度库。我们开发了一种增强型CLIP（eCLIP）方案，可将所需扩增减少约1，000倍，将丢弃的PCR重复读数减少约60%，同时保持单核苷酸结合分辨率。通过简化配对IgG和大小匹配的输入对照的生成，eCLIP提高了发现真实结合位点的特异性。我们在HepG 2和K562细胞中对73种不同的RBP进行了102次eCLIP实验（可在https://www.encodeproject.org上获得），证明eCLIP能够进行大规模和稳健的分析，扩增和样品要求与ChIP-seq相似。eCLIP能够对不同的RBP进行综合分析，以揭示因子特异性谱、CLIP的常见伪影和RBP活性的RNA中心观点。
As RNA binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNAs, binding site identification by UV-crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ~1,000-fold, decreasing discarded PCR duplicate reads by ~60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives of RBP activity.