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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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要蛋白质-DNA 相互作用控制对染色质中基因的访问并调节空间和时间特异性基因表达。这些相互作用对于细胞分化和细胞多样性的维持至关重要。蛋白质-DNA 相互作用的失调模式通常与疾病状态有关。接下来的应用- 一代表观基因组测序技术已经能够解卷积蛋白质-DNA相互作用单细胞水平。然而，定义蛋白质-DNA 中细胞间的异质性如何影响仍然具有挑战性。相互作用影响基因表达变异性。目前可用的蛋白质-DNA 联合分析方法单细胞的相互作用和基因表达需要费力的基因操作，受到的影响相对较低吞吐量，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。为了解决这些未满足的需求，我们建议开发 PRODIGE——一种用于联合无需遗传分析即可分析同一单细胞中的蛋白质-DNA 相互作用和基因表达操纵。它是通过可逆细胞固定、抗体引导染色质的战略整合来实现的标记、新型可变形和可降解的条形码水凝胶珠、液滴微流体以及下一步世代测序技术。在目标 1 中，我们将开发以下技术组件和分析管道：神童。我们将准备用于抗体引导的 Tn5 标记的蛋白 A-Tn5 转座体，制造用于单细胞 PRODIGE 测定的微流体装置，并合成可变形和用于单细胞条形码的可降解条形码水凝胶珠。在目标 2 中，我们将批量优化该方法，单细胞水平，并与其他独立的相比评估 PRODIGE（联合分析）的性能同行。在目标 3 中，我们将以 PRODIGE 为基准来对蛋白质进行联合单细胞分析的既定工具进行基准测试。 DNA 相互作用和转录组。拟议计划的成功实施将带来高通量工具，用于量化蛋白质-DNA 结合中细胞间异质性如何影响基因表达变异性，破译上游蛋白质-DNA 相互作用的细胞类型特异性模式及其转录输出，并确定调节细胞类型相关转录程序的蛋白质介导机制在异质细胞群中以及在不同的发育和/或疾病条件下。