Semi-permeable capsules for high-throughput single cell multi-omics

项目摘要 单细胞“多组学”代表了一套有前途的新兴工具，可以在细胞间建立关键联系。 转录程序及其潜在驱动因素，例如突变和信号通路的活性。 这些工具具有广泛的应用，以了解疾病的机制，组织发育， 再生该方法涉及每个细胞的转录组与其他细胞的转录组的同时分析。 测量，如DNA序列，表观遗传学和蛋白质组成。 单细胞基因组学的领先高通量范式具有严重影响其生物学特性的技术局限性。 它们在多路复用中的应用。目前只有少数全基因组测定以高细胞通量进行了多重化， 并且在复用时保持良好的灵敏度方面仍然存在技术挑战。一些联合 测量仍然没有得到证实，并且可能不可能用现有的方法来实现。 我们在这里开发了一种新的技术，用于使用亚纳升的单细胞多步生化分析 半渗透隔室（或“胶囊”）。胶囊应允许多个复杂的测量 单细胞目前还不可能，应该提高现有单细胞多组学数据的质量 这是因为它克服了现有平台的局限性。细胞在一个过程中被捕获在胶囊中 这种方法既便宜又快速，在那里它们可以被裂解并被条形码化以进行测序。与广泛使用的方法不同， 单细胞基因组学捕获液滴中的细胞，胶囊不是由油隔离：相反，它们交换 分子与其环境，这使得能够在处理单个 细胞裂解物。核酸被捕获在胶囊中而没有交联。胶囊也可以进行分类， 筛选和富集用于分析的细胞裂解物。 这项R21提案将使胶囊在单细胞多组学中得以实践。它植根于初步数据 在这里，我们展示了胶囊的定义技术要求。如果成功，这项工作将建立一个 强大的单细胞多模式检测新技术，克服了现有方法的局限性。在 这样做，我们将实施一种多组学分析，并将其应用于筛查和癌症表型分析， 将为实施其他高质量的多组学分析奠定基础。

Project summary Single cell “multi-omics” represents a promising set of emerging tools to establish crucial links between transcriptional programs and their underlying drivers such as mutations and the activity of signaling pathways. These tools have broad application to understand mechanisms of disease, tissue development and regeneration. The methods involve the simultaneous analysis of the transcriptome of each cell with other measurements such as DNA sequence, epigenetics and protein composition. The leading high-throughput paradigms for single cell genomics have technical limitations that severely impact their use in multiplexing. Only a few genome-wide assays have now been multiplexed at high cell throughput, and there remains a technical challenge in maintaining good sensitivity upon multiplexing. Some joint measurements remain undemonstrated and may be impossible to implement with existing methods. We develop here a novel technology for multi-step biochemical analysis of single cells using sub-nanoliter semi-permeable compartments (or “capsules”). Capsules should allow multiple complex measurements on single cells not currently possible, and should improve the quality of data from existing single cell multi-omic efforts because they overcome the limitations of existing platforms. Cells are captured in capsules in a process that is cheap and fast, where they may be lysed and barcoded for sequencing. Unlike widely-used methods for single cell genomics that capture cells in droplets, the capsules are not isolated by oil: instead they exchange molecules with their environment, which enables multiple reaction buffer exchanges while processing single cell lysates. Nucleic acids are trapped in capsules without cross-linking. Capsules can also be sorted to allow screening and enrichment of cell lysates for analysis. This R21 proposal will bring capsules to practice in single cell multi-omics. It is rooted in preliminary data where we demonstrate the defining technical requirements of capsules. If successful, this work will establish a powerful new technology for single cell multi-modal assays that overcomes limitations of existing methods. In doing so we will implement a multi-omic assay with application to screening and cancer phenotyping, and we will set the stage for implementing additional high quality multi-omic assays.