Determining chromatin-associated protein localization across the genome has provided insight into the functions of DNA-binding proteins and their connections to disease. However, established protocols requiring large quantities of cell or tissue samples currently limit applications for clinical and biomedical research in this field. Furthermore, most technologies have been optimized to assess abundant histone protein localization, prohibiting the investigation of nonhistone protein localization in low cell numbers. We recently described a protocol to profile chromatin-associated protein localization in as low as one cell: ultra-low-input cleavage under targets and release using nuclease (uliCUT&RUN). Optimized from chromatin immunocleavage and CUT&RUN, uliCUT&RUN is a tethered enzyme-based protocol that utilizes a combination of recombinant protein, antibody recognition and stringent purification to selectively target proteins of interest and isolate the associated DNA. Performed in native conditions, uliCUT&RUN profiles protein localization to chromatin with low input and high precision. Compared with other profiling technologies, uliCUT&RUN can determine nonhistone protein chromatin occupancies in low cell numbers, permitting the investigation into the molecular functions of a range of DNA-binding proteins within rare samples. From sample preparation to sequencing library submission, the uliCUT&RUN protocol takes <2 d to perform, with the accompanying data analysis timeline dependent on experience level.

确定染色质相关蛋白在基因组中的定位有助于深入了解 DNA 结合蛋白的功能及其与疾病的关系。然而，目前需要大量细胞或组织样本的既定方案限制了该领域临床和生物医学研究的应用。此外，大多数技术已经过优化以评估丰富的组蛋白定位，从而禁止在低细胞数量中研究非组蛋白定位。我们最近描述了一种在低至一个细胞中分析染色质相关蛋白定位的方案：在目标下进行超低输入切割并使用核酸酶释放（uliCUT&RUN）。uliCUT&RUN 是一种基于束缚酶的方案，对染色质免疫切割和 CUT&RUN 进行了优化，结合重组蛋白、抗体识别和严格纯化来选择性地靶向感兴趣的蛋白并分离相关 DNA。uliCUT&RUN 在天然条件下进行，以低输入和高精度分析蛋白质在染色质上的定位。与其他分析技术相比，uliCUT&RUN 可以确定低细胞数量中的非组蛋白蛋白染色质占据，从而可以研究稀有样品中一系列 DNA 结合蛋白的分子功能。从样品制备到测序文库提交，uliCUT&RUN 协议的执行时间不到 2 天，随附的数据分析时间表取决于经验水平。