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: 10569373
• 负责人: Allon Moshe Klein
• 金额: $ 20.13万
• 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/10569373
• 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; Plant Roots; Preparation; Process; Property; Proteins; RNA; Reaction; Reagent; Regenerative Medicine; Risk; Screening for cancer; Signal Pathway; Sorting - Cell Movement; Technology; Temperature; Tissue Sample; Tissues; Validation; Work; base; cancer cell; capsule; crosslink; data quality; design; genome-wide; human tissue; improved; interest; multimodality; multiple omics; multiplex assay; nanolitre; new technology; novel; programs; screening; single cell analysis; single cell technology; single-cell RNA sequencing; success; 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。 该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 它们在多重分析中的应用现在只有少数全基因组检测能够以高细胞通量进行多重分析， 并且在复用时保持良好的灵敏度仍然存在技术挑战。 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”）应允许进行多种复杂的测量。 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 因为它们克服了现有努力平台的局限性，在一个过程中将细胞捕获在胶囊中。 与广泛使用的测序方法不同，这种方法便宜且快速，可以将它们裂解并打上条形码。 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 核酸被捕获在胶囊中而无需交联。 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中，它植根于初步数据。 我们将展示胶囊的定义技术要求。如果成功，这项工作将建立一个明确的技术要求。 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。 为此，我们将实施应用于筛查和癌症表型分析的多组学测定，并且我们 将为实施额外的高质量多组学分析奠定基础。