Abstract：
Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles.
摘要：
染色质内源性切割与高通量测序（ChEC-seq）是一种蛋白质-DNA相互作用的分析方法，可以在体内检测结合位置，不需要抗体或固定，并提供接近核苷酸分辨率的全基因组覆盖。该方法的核心是目标蛋白的MNase融合，这允许它，当被钙暴露触发时，在其结合位点切割DNA并产生小的DNA片段，这些片段可以很容易地与基因组的其余部分分离并测序。自原始协议以来的改进增加了易用性，降低了成本,并乘以该方法的吞吐量，以实现ChIP-seq等传统方法无法实现的实验规模和分辨率。该方法描述了从MNase标记的酵母菌株的初始创建和验证的每个步骤，通过ChECMNase活化和小片段纯化程序进行测序文库制备。它还简要介绍了创建有意义的全基因组结合谱所需的生物信息学步骤。
