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

用于高通量单细胞多组学的半透胶囊

• 批准号: 10698044
• 负责人: Allon Moshe Klein
• 金额: $ 24.58万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-09 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10698044
• 关键词: ATAC-seq; Address; Adsorption; Bar Codes; Biochemical; Biochemical Reaction; Biological Assay; Biomedical Research; Buffers; Cell Aggregation; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Sequence; DNA Sequence Alteration; Data; Development; Disease; Engineering; Environment; Epigenetic Process; Event; Fibrosis; Genetic Transcription; Genomic DNA; Genomics; Genotype; Goals; Grant; In Situ; Joints; Libraries; Link; Malignant Neoplasms; Measurement; Membrane; Messenger RNA; Methods; Microfluidics; Molecular Analysis; Mutation; National Human Genome Research Institute; Natural regeneration; Nucleic Acids; Oils; Performance; Permeability; Phenotype; Preparation; Process; Property; Proteins; RNA; Reaction; Reagent; Regenerative Medicine; Risk; Signal Pathway; Sorting; Technology; Temperature; Tissue Sample; Tissues; Validation; Work; cancer cell; capsule; crosslink; data quality; design; genome-wide; human tissue; improved; interest; multimodality; multiple omics; multiplex assay; nano; nanolitre; new technology; novel; programs; resilience; screening; single cell analysis; single cell technology; single-cell RNA sequencing; success; technology platform; tool; transcriptome; transcriptome sequencing

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.

项目摘要 单个单元“多词”代表了一套有希望的新兴工具，可以在之间建立关键的联系 转录程序及其基础驱动因素，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，组织发育和 再生。该方法涉及对每个单元的转录组的简单分析 测量如DNA序列，表观遗传学和蛋白质组成。 领先的单细胞基因组学高通量范式具有严重影响的技术局限性 它们用于多路复用。现在，只有少数几个全基因组测定在高细胞吞吐量下多发 而且，在多路复用时保持良好的灵敏度仍然存在技术挑战。一些关节 测量值仍然不符合，可能无法使用现有方法实施。 我们在这里开发了一种新型技术，用于使用子纳米素的单细胞多步生物化学分析 半渗透室（或“胶囊”）。胶囊应允许对 当前无法使用单个单元格，应提高现有单细胞多OMIC的数据质量 努力是因为他们克服了现有平台的局限性。在一个过程中将细胞捕获在胶囊中 这是便宜又快速的，可以将它们推理和条形码进行测序。与广泛使用的方法不同 单细胞基因组学捕获细胞在液滴中的单细胞基因组学，胶囊不是通过油隔离的：相反，它们交换 与其环境的分子，可以在处理单一时进行多个反应缓冲液交换 细胞裂解物。核酸被困在没有交联的胶囊中。胶囊也可以分类以允许 筛选和富集细胞裂解物以进行分析。 该R21提案将使胶囊在单细胞多摩学中进行练习。它源于初步数据 我们证明了胶囊的定义技术要求。如果成功，这项工作将建立 用于克服现有方法限制的单细胞多模式测定的强大新技术。在 这样做，我们将实施一项多运动评估，并应用于筛查和癌症表型，我们 将为实施其他高质量的多摩变测定法奠定基础。