Department of Molecular Genetics, Weizmann Institute, Rehovot, Israel.
School of Science, Constructor University, Bremen, Germany.
Methods Mol Biol. 2024;2846:263-283. doi: 10.1007/978-1-0716-4071-5_16.
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.
染色质内源性切割结合高通量测序（ChEC-seq）是一种蛋白质-DNA 相互作用的分析方法，可在体内检测结合位置，不需要抗体或固定，并且可以在近核苷酸分辨率下提供全基因组覆盖。该方法的核心是目标蛋白的 MNase 融合，当受到钙暴露的触发时，它可以在其结合位点切割 DNA，并生成可与基因组其余部分轻松分离和测序的小 DNA 片段。自原始方案以来的改进提高了这种方法的易用性、降低了成本，并提高了通量，使实验的规模和分辨率达到了传统方法（如 ChIP-seq）所无法达到的水平。本方法描述了从最初创建和验证带有 MNase 标签的酵母菌株，到 ChEC MNase 激活和小片段纯化步骤，再到测序文库制备的每个步骤。它还简要介绍了创建有意义的全基因组结合图谱所需的生物信息学步骤。
