Droplet Microfluidic Platform for Ultralow Input Epigenetics

 描述（由申请人提供）：表观基因组分析在开发治疗癌症的个性化策略中发挥着越来越重要的作用。然而，将基本的表观基因组学见解转化为临床是充满挑战的。以翻译后组蛋白修饰的研究为例，它可以促进或抑制悬垂DNA序列的转录。对这些至关重要的相互作用的分析是基因组学和蛋白质组学的桥梁，并在临床环境中提出了重大挑战。染色质免疫沉淀（ChIP）是分析蛋白质-DNA相互作用的首选方法，基本方法包括染色质片段化、使用抗体和磁珠分离修饰的蛋白质以及随后通过qPCR或测序分析相关DNA。虽然听起来很简单，但典型的ChIP工作流程涉及约30个步骤，需要4天以上，并需要106-107个细胞作为输入。这些要求大大限制了ChIP在临床环境中的适用性，特别是当最小样本可用时，例如在肿瘤活检、干细胞或循环肿瘤细胞的分析中。微流体装置提供了许多优于传统宏观尺度方法的有吸引力的益处，包括减少的体积要求、并行化能力和自动化操作，这使得它们特别适合于样品约束的表观遗传分析。最近的一些报告表明，微流体支持的ChIP分析有很大的机会;然而，仍有相当大的进一步改进的空间。我们建议开发一种功能强大、多功能的、基于液滴微流体的、纳升级染色质免疫捕获（nChIC）平台，适用于个性化医学应用。液滴微流体提供了几个好处，包括快速，受控和有效的流体处理，以及处理可变样品尺寸的能力，因为设备可以通过简单地操作更长的时间来容纳更大的样品。我们的nChIC平台将把ChIP工作流程中的每一个主要步骤整合到自动化设备中，包括细胞裂解、染色质消化、免疫捕获和DNA纯化。重要的是，这些过程将在单细胞水平上进行，这有望为表观基因组肿瘤异质性提供独特的见解。除了单细胞之外，前所未有的处理可变输入样本的能力也将促进对传统ChIP检测的稳健验证，以证明广泛的基因组覆盖。综上所述，我们认为nChIC平台将是一个强大的新工具，能够将表观基因组学的见解转化为个性化的癌症治疗。

 DESCRIPTION (provided by applicant): Epigenomic analyses are playing increasingly prominent roles in the development of personalized strategies for treating cancer. However, the translation of fundamental epigenomic insight to the clinic is wrought with challenges. Take for example the study of post-translational histone protein modifications, which can serve to either promote or repress the transcription of pendant DNA sequences. Analyses of these critically important interactions bridge genomics and proteomics and present significant challenges in the clinical setting. Chromatin immunoprecipitation (ChIP) is the method of choice for analyzing protein-DNA interactions and the basic method involves fragmentation of chromatin, separation of modified proteins using antibodies and magnetic beads, and subsequent analysis of the associated DNA by qPCR or sequencing. While sounding simple, a typical ChIP workflow involves ~30 steps, takes 4+ days, and requires 106-107 cells as input. These requirements significantly limit the applicability of ChIP in a clinical setting-particularly when minimal sampl is available, such as in the analysis of tumor biopsies, stem cells, or circulating tumor cells. Microfluidic devices offer many attractive benefits over traditional macro-scale methods including reduced volume requirements, parallelization capability, and automated operation, which make them particularly well- suited to sample-constrained epigenetic analyses. A handful of recent reports suggest a substantial opportunity for microfluidically-enabled ChIP analyses; however, there is still considerable room for further improvement. We propose to develop a powerful and versatile, droplet microfluidics-based, nanoliter-scale Chromatin ImmunoCapture (nChIC) platform suitable for individualized medicine applications. Droplet microfluidics offer several benefits, including rapid, controlled, and efficient fluid handling, and the capacity to handle variable sample sizes, since devices can accommodate larger samples by simply operating for longer periods of time. Our nChIC platform will incorporate every major step in the ChIP workflow into an automated device, including cell lysis, chromatin digestion, immunocapture, and DNA purification. Importantly, these processes will be carried out at the single cell level, which promises to provide unique insights into epigenomic tumor heterogeneity. Beyond single cells, the unprecedented ability to handle samples of variable input will also facilitate robust validation against traditional ChIP assays to demonstrate broad genomic coverage. Taken together, we feel that the nChIC platform will be a powerful new tool that enables the translation of epigenomic insight into individualized cancer treatment at the point of care.