Simplified ChIP-exo assays
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Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and its ultra-high resolution cousin ChIP-exo are methods that identify where proteins bind along any genome in vivo. ChIP-exo achieves near-base pair resolution by creating exonuclease stop sites just 5â to where formaldehyde-induced protein-DNA cross-links occur. Whereas construction of ChIP genomic libraries is straightforward and widely adopted for ChIP-seq, ChIP-exo is technically more involved which has resulted in limited adoption. Here we describe multiple ChIP-exo protocols, each with use-specific advantages and limitations. The new versions are greatly simplified through removal of multiple enzymatic steps. This is achieved in part through the use of Tn5 tagmentation and/or single-stranded DNA ligation. The result is greater library yields, lower processing time, and lower cost. A similar streamlined approach was developed for ChIP-seq, called ChIP-seq 1-step, where library construction is achieved in one-step.
染色质免疫共沉淀结合深度测序（Chromatin immunoprecipitation followed by deep sequencing, ChIP-seq）及其超高分辨率同类技术ChIP-exo，均可在体内全基因组范围内精准定位蛋白质的结合位点。ChIP-exo通过在甲醛诱导的蛋白质-DNA交联位点的5'端处创建核酸外切酶终止位点，实现了接近单碱基对的分辨率。尽管ChIP-seq的基因组文库构建流程简单易行且已被广泛应用，但ChIP-exo的技术流程更为复杂，这导致其应用范围受限。本文详述了多种ChIP-exo实验方案，每种方案均具备对应应用场景下的优势与局限性。新版本方案通过移除多个酶促反应步骤，大幅简化了实验流程，这一优化部分得益于使用了Tn5转座标签化（Tn5 tagmentation）技术以及/或单链DNA连接技术。最终实现了更高的文库产出量、更短的实验处理时长与更低的实验成本。针对ChIP-seq也开发了类似的简化流程，被命名为一步法ChIP-seq（ChIP-seq 1-step），其文库构建可通过单一步骤完成。
