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

项目总结 单细胞“多组学”代表了一套很有前途的新兴工具，可以在 转录程序及其潜在的驱动因素，如突变和信号通路的活性。 这些工具在了解疾病的机制、组织发育和 再生。这些方法包括同时分析每个细胞和其他细胞的转录组。 DNA序列、表观遗传学和蛋白质组成等测量。 单细胞基因组学的领先高通量范例具有严重影响 它们在多路传输中的使用。现在只有几种全基因组分析在高细胞吞吐量下被多路复用， 而且，在多路传输时保持良好的灵敏度仍然是一个技术挑战。一些接头 测量仍然没有得到证实，可能不可能用现有的方法来实施。 我们在这里开发了一种利用亚纳升进行单细胞多步生化分析的新技术 半透水舱(或“胶囊”)。胶囊应允许多次复杂的测量 单细胞目前还不可能，应该可以提高现有单细胞多组体的数据质量 这些努力是因为它们克服了现有平台的限制。在一个过程中，细胞被捕获在胶囊中 这是廉价和快速的，在那里它们可以被裂解并进行条形码测序。与广泛使用的方法不同 单细胞基因组学捕捉水滴中的细胞，这些胶囊不是通过石油分离的：相反，它们交换 分子及其环境，这使得在处理单个反应时能够进行多个反应缓冲区交换 细胞裂解物。核酸被困在胶囊中，没有交联物。胶囊也可以分类，以允许 用于分析的细胞裂解产物的筛选和浓缩。 这项R21提案将使胶囊在单细胞多组学中得到实践。它植根于初步数据 在这里我们演示了DefiNing胶囊的技术要求。如果成功，这项工作将建立一个 强大的单细胞多模式分析新技术，克服了现有方法的局限性。在……里面 为此，我们将实施多组分析，并应用于筛查和癌症表型鉴定，我们 将为实施额外的高质量多组体分析奠定基础。

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.