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

项目概要 单细胞“多组学”代表了一组有前景的新兴工具，可在细胞之间建立关键联系 转录程序及其潜在驱动因素，例如突变和信号通路的活动。 这些工具在理解疾病、组织发育和疾病机制方面具有广泛的应用。 再生。这些方法涉及同时分析每个细胞与其他细胞的转录组 DNA 序列、表观遗传学和蛋白质组成等测量。 单细胞基因组学领先的高通量范例存在严重影响的技术限制 它们在多路复用中的用途。现在只有少数全基因组检测能够以高细胞通量进行多重检测， 并且在复用时保持良好的灵敏度仍然存在技术挑战。一些联合 测量结果尚未得到证实，并且可能无法用现有方法实施。 我们在这里开发了一种使用亚纳升对单细胞进行多步生化分析的新技术 半渗透隔室（或“胶囊”）。胶囊应允许对多个复杂的测量 单细胞目前不可能，应该提高现有单细胞多组学的数据质量 努力是因为它们克服了现有平台的局限性。在一个过程中细胞被捕获在胶囊中 这是便宜且快速的，可以将它们裂解并打上条形码以进行测序。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，胶囊不会被油隔离：相反，它们会交换 分子与其环境，这使得在处理单个反应缓冲液时能够交换多个反应缓冲液 细胞裂解物。核酸被捕获在胶囊中而不发生交联。还可以对胶囊进行分类，以允许 筛选和富集细胞裂解物以供分析。 R21 提案将把胶囊应用于单细胞多组学实践。它植根于初步数据 我们展示了胶囊的定义技术要求。如果成功的话，这项工作将建立一个 用于单细胞多模式测定的强大新技术，克服了现有方法的局限性。在 为此，我们将实施多组学测定，并将其应用于筛查和癌症表型分析，并且我们 将为实施额外的高质量多组学检测奠定基础。

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.