PRODIGE: A high-throughput tool for joint profiling of protein-DNA interactions and gene expression in single cells

PRODIGE：一种高通量工具，用于联合分析单细胞中蛋白质-DNA 相互作用和基因表达

• 批准号: 10492769
• 负责人: Wei Wei
• 金额: $ 22.88万
• 依托单位: INSTITUTE FOR SYSTEMS BIOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10492769
• 关键词: Address; Antibodies; Bar Codes; Benchmarking; Binding; Biological Assay; Cell Differentiation process; Cell Line; Cells; Chromatin; Clinical; Complex; DNA; DNA-Binding Proteins; DNA-Protein Interaction; Data; Data Set; Development; Disease; Drops; Epigenetic Process; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Heterogeneity; Histones; Hydrogels; Individual; Joints; Libraries; Maintenance; Maps; Measurement; Measures; Mediating; Messenger RNA; Methods; Microfluidic Microchips; Microfluidics; Modality; Modeling; Molecular; Output; Pattern; Performance; Phenotype; Play; Poly A; Population; Post-Translational Protein Processing; Process; Protein Analysis; Proteins; Protocols documentation; RNA; Regulator Genes; Reporting; Reproducibility; Research Personnel; Resolution; Role; Sampling; System; Techniques; Technology; Testing; Tissues; cancer cell; cell fixation; cell type; computational pipelines; data analysis pipeline; droplet sequencing; epigenomics; genetic manipulation; genome-wide; histone modification; insight; interest; methylome; multiple omics; next generation; next generation sequencing; novel; programs; protein profiling; single-cell RNA sequencing; tool; transcription factor; transcriptome; Address; Antibodies; Bar Codes; Benchmarking; Binding; Biological Assay; Cell Differentiation process; Cell Line; Cells; Chromatin; Clinical; Complex; DNA; DNA-Binding Proteins; DNA-Protein Interaction; Data; Data Set; Development; Disease; Drops; Epigenetic Process; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Heterogeneity; Histones; Hydrogels; Individual; Joints; Libraries; Maintenance; Maps; Measurement; Measures; Mediating; Messenger RNA; Methods; Microfluidic Microchips; Microfluidics; Modality; Modeling; Molecular; Output; Pattern; Performance; Phenotype; Play; Poly A; Population; Post-Translational Protein Processing; Process; Protein Analysis; Proteins; Protocols documentation; RNA; Regulator Genes; Reporting; Reproducibility; Research Personnel; Resolution; Role; Sampling; System; Techniques; Technology; Testing; Tissues; cancer cell; cell fixation; cell type; computational pipelines; data analysis pipeline; droplet sequencing; epigenomics; genetic manipulation; genome-wide; histone modification; insight; interest; methylome; multiple omics; next generation; next generation sequencing; novel; programs; protein profiling; single-cell RNA sequencing; tool; transcription factor; transcriptome

PROJECT SUMMARY/ABSTRACT Protein-DNA interactions control access to genes in chromatins and regulate the spatial- and temporal-specific gene expression. These interactions are crucial for cell differentiation and the maintenance of cellular diversity. Dysregulated patterns of protein-DNA interactions are often associated with disease states. Applications of next- generation epigenomic sequencing techniques have enabled deconvolution of protein-DNA interactions at single-cell level. However, it remains challenging to define how cell-to-cell heterogeneity in protein-DNA interactions impacts gene expression variability. Currently available methods for joint profiling protein-DNA interactions and gene expression from single cells require laborious genetic manipulation, suffer relatively low throughput, and are impossible to specifically map proteins with post-translational modifications, such as histone marks. To address these unmet needs, we propose to develop PRODIGE – a high-throughput tool for joint profiling of protein-DNA interactions and gene expression in the same single cells without the need of genetic manipulations. It is enabled by a strategic integration of reversible cell fixation, antibody-guided chromatin tagmentation, novel deformable and degradable barcoded hydrogel beads, droplet microfluidics, and next- generation sequencing technology. In Aim 1, we will develop technical components and analytical pipelines of PRODIGE. We will prepare protein A-Tn5 transposome for antibody-guided Tn5 tagmentation, fabricate microfluidic devices for single-cell PRODIGE assay, and synthesize a barcoded library of deformable and degradable barcoded hydrogel beads for single-cell barcoding. In Aim 2, we will optimize the method in bulk and single-cell levels, and assess the performance of PRODIGE (joint analysis) in comparison to other stand-alone counterparts. In Aim 3, we will benchmark PRODIGE to established tools for joint single-cell profiling of protein- DNA interactions and transcriptome. Successful implementation of the proposed program will deliver a high- throughput tool to quantify how cell-to-cell heterogeneity in protein-DNA binding influences gene expression variability, decipher cell-type-specific patterns of upstream protein-DNA interactions and their transcriptional outputs, and identify protein-mediated mechanisms that regulate cell-type-associated transcriptional programs in heterogeneous cell populations and under different developmental and/or disease conditions.

项目摘要/摘要 蛋白-DNA相互作用控制染色蛋白中对基因的访问，并调节空间和临时特异性 基因表达。这些相互作用对于细胞分化和维持细胞多样性至关重要。 蛋白-DNA相互作用的失调模式通常与疾病状态有关。接下来的申请 产生的表观基因组测序技术已实现了在蛋白质-DNA相互作用的反卷积 单细胞水平。但是，定义细胞到细胞的异质性在蛋白质-DNA中如何保持挑战 相互作用会影响基因表达变异性。当前可用的联合分析蛋白-DNA方法 来自单细胞的相互作用和基因表达需要实验室基因操纵，遭受相对较低的 吞吐量，并且不可能特异性地绘制具有翻译后修饰的蛋白质，例如组蛋白 标记。为了满足这些未满足的需求，我们建议开发Prodige - 一种用于关节的高通量工具 蛋白-DNA相互作用和基因表达在同一单个细胞中的分析而无需通用 操纵。通过可逆细胞固定，抗体引导的染色质的战略整合来启用它 标签，新颖的可变形和可降解的条形水凝胶珠，液滴微流体和下一步 生成测序技术。在AIM 1中，我们将开发技术组件和分析管道 prodige。我们将准备蛋白A-TN5转座体用于抗体引导的TN5标记，制造 用于单细胞生产分析的微流体设备，并合成一个可变形和的条形码库 可降解的条形码水凝胶珠，用于单细胞条形码。在AIM 2中，我们将优化批量和 与其他独立的单细胞水平，并评估体力物的性能（联合分析） 同行。在AIM 3中，我们将基准为建立的工具基准，用于蛋白质的联合单细胞分析 DNA相互作用和转录组。拟议计划的成功实施将提供高级 量化工具以量化蛋白质-DNA结合中细胞到细胞的异质性如何影响基因表达 上游蛋白-DNA相互作用及其转录的变异性，解密细胞类型特异性模式 输出并确定调节细胞类型相关转录程序的蛋白质介导的机制 在异质细胞群体以及不同的发育和/或疾病状况下。