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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：
10303542
负责人：
Wei Wei
金额：
$ 27.45万
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 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10303542
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.

项目总结/摘要蛋白质-DNA相互作用控制染色质中基因的进入，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维持细胞多样性至关重要。蛋白质-DNA相互作用的失调模式通常与疾病状态相关。下一个应用- 第二代表观基因组测序技术使得蛋白质-DNA相互作用的解卷积成为可能，单细胞水平。然而，它仍然具有挑战性，以确定如何细胞间异质性蛋白质-DNA 相互作用影响基因表达变异性。目前可用的联合分析蛋白质-DNA的方法来自单细胞的相互作用和基因表达需要费力的遗传操作，并且不可能特异性地映射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-一种高通量的联合检测工具，蛋白质-DNA相互作用和基因表达谱在同一个单细胞中，而不需要遗传分析。操纵它是通过可逆的细胞固定，抗体引导的染色质标签片段化、新型可变形和可降解的条形码化水凝胶珠、液滴微流体，以及接下来的代测序技术。在目标1中，我们将开发技术组件和分析管道， PRODIGE.我们将制备蛋白A-Tn5转座体用于抗体引导的Tn5标签片段化，用于单细胞PRODIGE测定的微流控装置，并合成可变形和可降解的条形码文库。用于单细胞条形码化的可降解条形码化水凝胶珠。在目标2中，我们将批量优化该方法，单细胞水平，并评估PRODIGE（联合分析）的性能与其他独立同行在目标3中，我们将对PRODIGE进行基准测试，以建立用于蛋白质的联合单细胞分析的工具， DNA相互作用和转录组。该计划的成功实施将带来巨大的... 一种通量工具，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可变性，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，并确定蛋白质介导的机制，调节细胞类型相关的转录程序在异质细胞群体中以及在不同的发育和/或疾病条件下。