High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states
High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states
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DOI：
10.1038/nprot.2013.023
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发表时间：
2013-03-01
期刊：
影响因子：
14.8
通讯作者：
Amit, Ido
中科院分区：
文献类型：
--
作者：
Blecher-Gonen, Ronnie;Barnett-Itzhaki, Zohar;Amit, Ido
蛋白质与DNA元件的动态结合调节基因组功能和细胞命运。尽管绘制体内蛋白质- dna相互作用图谱的方法对基因组研究的各个方面都变得至关重要，但它们既费力又昂贵，因此限制了进展。在这里，我们提出了一种使用高通量染色质免疫沉淀（HT-ChIP）方法绘制体内蛋白质- dna相互作用的方案。通过使用顺磁珠，我们简化了整个ChIP和索引库构建过程：样品转移和损失最小化，并且消除了手动劳动密集型程序（如洗涤，凝胶提取和DNA沉淀）的需要。所有这些都可以实现全自动、低成本和高灵敏度的96孔芯片测序（ChIP-seq）。样品制备需要3天从培养细胞到汇集库。与以前的方法相比，HT-ChIP更适合于大规模的体内研究，特别是那些测量大量不同染色质修饰/转录因子或多重扰动的动力学的研究。
Dynamic protein binding to DNA elements regulates genome function and cell fate. Although methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research, they are laborious and costly, thereby limiting progress. Here we present a protocol for mapping in vivo protein-DNA interactions using a high-throughput chromatin immunoprecipitation (HT-ChIP) approach. By using paramagnetic beads, we streamline the entire ChIP and indexed library construction process: sample transfer and loss is minimized and the need for manually labor-intensive procedures such as washes, gel extraction and DNA precipitation is eliminated. All of this allows for fully automated, cost effective and highly sensitive 96-well ChIP sequencing (ChIP-seq). Sample preparation takes 3 d from cultured cells to pooled libraries. Compared with previous methods, HT-ChIP is more suitable for large-scale in vivo studies, specifically those measuring the dynamics of a large number of different chromatin modifications/transcription factors or multiple perturbations.