Simplified ChIP-exo assays

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 5’ 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）及其超高分辨率同系技术ChIP-exo，均为可在体内定位蛋白质沿任意基因组结合位点的实验方法。ChIP-exo通过在甲醛诱导的蛋白质-DNA交联位点的5'端附近构建核酸外切酶终止位点，实现了近单碱基分辨率。相较于ChIP-seq建库流程简单易行且已被广泛应用，ChIP-exo的技术操作更为繁琐，这导致其应用范围较为有限。本文介绍了多款ChIP-exo实验方案，各方案均具备特定的适用优势与局限。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，该优化部分借助Tn5转座酶标签化（Tn5 tagmentation）和/或单链DNA连接反应（single-stranded DNA ligation）得以实现。最终可获得更高的文库产出量、更短的实验处理时长以及更低的实验成本。针对ChIP-seq也开发了类似的简化流程，即所谓的“ChIP-seq 一步法（ChIP-seq 1-step）”，其建库过程可在单一步骤内完成。