Teste Bruno, Champ Jerome, Londono-Vallejo Arturo, Descroix Stéphanie, Malaquin Laurent, Viovy Jean-Louis, Draskovic Irena, Mottet Guillaume
Institut Curie, PSL Research University, CNRS UMR 168, 6 Rue Calvin, 75005 Paris, France.
CNRS, UMR 3244, Telomeres and Cancer Laboratory, Institut Curie, PSL Research University, F-75248, Paris, France and CNRS, UMR3244, Sorbonne Universités, UPMC Univ Paris 06, 75005, Paris, France.
Lab Chip. 2017 Jan 31;17(3):530-537. doi: 10.1039/c6lc01535b.
Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.
基因组织受DNA与组蛋白相互作用的调控，这些蛋白质的差异修饰是基因调控的基本机制。组蛋白修饰主要通过染色质免疫沉淀（ChIP）分析来研究，然而传统的ChIP方法耗时、费力，且需要大量细胞。在此，我们首次报道了基于微流控平台开发的液滴ChIP技术，该平台结合了纳升级液滴、磁珠（MB）和磁镊（MT）。液滴方法实现了分隔并改善了混合效果，同时在集成工作流程中减少了样品和试剂的消耗。将嫁接到磁珠上的抗组蛋白抗体用作固相支持物，以捕获目标染色质并将其从一个液滴转移到另一个液滴，从而进行染色质免疫沉淀、洗涤、洗脱和DNA纯化。我们设计了一种新的ChIP方案，用于研究在基因激活或抑制中具有已知作用的四种不同类型的修饰组蛋白。我们将这种新的液滴ChIP分析方法与传统方法进行了性能评估比较。所提出的技术将分析时间从几天大幅缩短至7小时，简化了ChIP方案，将所需细胞数量减少了100倍，同时保持了高度的灵敏度和特异性。因此，这种基于液滴的ChIP分析代表了一种新的、极具优势且便捷的表观遗传学分析方法。
