In situ Chromatin Interaction Analysis Using Paired-End Tag Sequencing.
In situ Chromatin Interaction Analysis Using Paired-End Tag Sequencing.
复制标题
DOI：
10.1002/cpz1.174
复制
发表时间：
2021-08
期刊：
影响因子：
--
通讯作者：
Ruan Y
中科院分区：
文献类型：
--
作者：
Wang P;Feng Y;Zhu K;Chai H;Chang YT;Yang X;Liu X;Shen C;Gega E;Lee B;Kim M;Ruan X;Ruan Y
使用配对末端标签测序（ChIA-PET）进行染色质相互作用分析是一种确定蛋白质介导的染色质相互作用的方法。然而，它的一个限制是每次实验需要1亿个细胞，这阻碍了它在生物医学研究中的广泛应用，特别是在难以从稀有样本中获得大量细胞的研究中。为了减少所需的输入细胞数，同时保持高数据质量，我们开发了原位china - pet协议，只需100万个细胞。在这里，我们描述了从培养细胞中进行原位ChIA-PET的详细步骤，包括样品制备和数据生成的实验方案，以及使用ChIA-PIPE管道进行数据处理和可视化的计算方案。由于该方案大大简化了实验程序，减少了连接噪声，并且与以前版本的china - pet方案相比减少了所需的输入细胞，因此可用于为广泛的人和小鼠原代细胞生成由各种蛋白质因子介导的高分辨率染色质接触图。
Chromatin Interaction Analysis Using Paired‐End Tag Sequencing (ChIA-PET) is an established method to map protein-mediated chromatin interactions. A limitation, however, is that it requires a hundred million cells per experiment, which hampers its broad application in biomedical research, particularly in studies in which it is intractable to obtain a large number of cells from rare samples. To reduce the required input cell number while retaining high data quality, we developed an in situ ChIA-PET protocol, which requires as few as 1 million cells. Here, we describe the detailed step-by-step procedures for performing in situ ChIA-PET from cultured cells, including both experimental protocols for sample preparation and data generation, and a computational protocol for data processing and visualization using the ChIA-PIPE pipeline. As the protocol significantly simplifies the experimental procedure, reduces the ligation noise, and decreases the required input cells compared to previous versions of ChIA-PET protocols, it can be applied to generate high-resolution chromatin contact maps mediated by various protein factors for a wide range of human and mouse primary cells.