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

染色质免疫沉淀（ChIP）与测序（ChIP-seq）结合用于体外蛋白质-DNA相互作用的研究，其基础是通过下一代测序（NGS）对ChIP获得的感兴趣的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。缩小芯片实验的规模和开发同时进行芯片反应的平台正在成为一个流行的趋势，因为最小化实验的持续时间和成本是很重要的。目前的平台要么依赖于微滴板格式，并与大的反应体积和初始细胞数相关联，要么依赖于微流控原理，但与高通量不兼容。我们最近开发了一种基于超亲水性-超疏水性（SL-SH）模式的小型活细胞高通量筛选平台，称为微滴阵列（DMA）。DMA平台能够在标准显微镜载玻片格式上筛选数百到数千个因子，体积范围从3到80 nL。这样的屏幕可以并行制作，而不需要移液每个单独的点。在这里，我建议调整DMA平台进行高通量ChIP-seq实验，最初只有100到1000个活细胞。该项目的主要目标是开发和优化DMA平台上的高度并行ChIP-seq协议。此外，这种新方法将通过在DMA上进行概念验证筛选来评估高通量应用。在这种情况下，细胞将在DMA平台上被引入不同的刺激，并行ChIP-seq分析将在液滴本身中进行，以区分处理细胞群体的最终表观遗传状态。利用DMA平台进行高通量ChIP反应优于当前技术，因为它可以大幅减少反应体积，同时确保更高的通量DMA技术还具有将高通量筛选（将细胞引入化合物库）和ChIP分析结合起来的潜力，作为在单个液滴中执行的读出，这一过程在使用任何当前平台时都是不可能的。

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.