ChIPseq-on-Chip Droplet-Microarray as a miniaturized high throughput platform for parallel ChIP-seq experiments

染色质免疫沉淀（ChIP）结合测序（ChIP-seq）用于研究体外蛋白质-DNA相互作用，并基于通过下一代测序（NGS）对ChIP获得的目标DNA片段进行直接测序。ChIP-seq技术被广泛用于从根本上评估染色质结构和功能，以及设计和开发新的表观遗传药物。缩小ChIP实验和开发同时进行ChIP反应的平台正在成为一种流行趋势，因为最大限度地减少实验的持续时间和成本非常重要。目前的平台要么依赖于微量滴定板格式并与大反应体积和初始细胞数量相关，要么依赖于尚未与高通量兼容的微流体原理。我们最近开发了一种基于超疏水-超疏水（SL-SH）模式的小型化活细胞高通量筛选平台，称为微滴微阵列（DMA）。DMA平台可以在3至80 nL的标准显微镜载玻片格式上筛选数百至数千个因子。这样的筛选可以并行进行，而不需要移液每个单独的斑点。在这里，我建议调整DMA平台，以进行高通量ChIP-seq实验，最初只有100到1000个活细胞。该项目的主要目标是在DMA平台上开发和优化高度并行的ChIP-seq协议。此外，这种新的方法将通过对DMA的概念验证筛选进行高通量应用评估。在这种情况下，细胞将被引入DMA平台上的不同刺激物，并且平行的ChIP-seq分析将在液滴本身中进行，以区分经处理的细胞群体中的最终表观遗传状态。利用DMA平台进行高通量ChIP反应上级当前技术，因为它能够显著减少反应体积，同时确保更高通量的DMA技术还具有将高通量筛选（将细胞引入化合物文库）和ChIP分析结合为在单个液滴中进行的读出的潜力，这是在使用任何当前平台时都不可能实现的程序。

Chromatin immunoprecipitation (ChIP) in combination with sequencing (ChIP-seq) is used for investigation of protein-DNA interactions in vitro and based on direct sequencing of DNA fragments of interest obtained by ChIP via next generation sequencing (NGS). ChIP-seq technology is widely used to fundamentally assess chromatin structure and function, as well as to design and develop new epigenetic drugs. Scaling down ChIP experiments and developing platforms to carry out simultaneous ChIP reactions are becoming a popular trend, because it is important to minimize the experiment's duration and cost. The current platforms rely either on the microtiter plate format and associated with large reaction volumes and initial cell numbers, or on the microfluidic principle not yet compatible with high-throughput. We recently developed a miniaturized platform for live cell high-throughput screening based on superhydrophilic-superhydrophobic (SL-SH) patterning called Droplet-Microarray (DMA). The DMA platform enables the screening of hundreds to thousands of factors on a standard microscope slide format in volumes ranging from 3 to 80 nL. Such screens can be made in parallel without the need to pipet each individual spot. Here I propose to adapt the DMA platform to conduct high-throughput ChIP-seq experiments starting initially with as few as 100 to 1000 live cells. The main goal of this project is to develop and optimize protocols for highly parallel ChIP-seq on the DMA platform. In addition, this novel methodology will be evaluated for high-throughput applications via proof-of-concept screening on DMA. In this case cells will be introduced to different stimuli on the DMA platform and parallel ChIP-seq analysis will take place in the droplets themselves to distinguish between the final epigenetic states in treated cell populations. Utilizing the DMA platform for high-throughput ChIP reactions is superior to current technologies because it enables a dramatic reduction in reaction volumes while ensuring higher throughput DMA technology also has the potential to combine high throughput screening (introducing cells to a compound library) and ChiP analysis as a read-out performed in a single droplet, a procedure that is impossible when working with any of the current platforms.