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

项目摘要/摘要 蛋白质-DNA相互作用控制着对染色质中基因的访问，并调节空间和时间上的特异性 基因表达。这些相互作用对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有关。NEXT的应用- 世代表观基因组测序技术实现了蛋白质-DNA相互作用的去卷积 单细胞水平。然而，如何定义蛋白质DNA中细胞间的异质性仍然具有挑战性。 相互作用影响基因表达的可变性。目前可用的蛋白质-DNA联合图谱方法 单细胞的相互作用和基因表达需要费力的遗传操作，受到的影响相对较低 吞吐量，并且不可能特异地定位具有翻译后修饰的蛋白质，如组蛋白 马克。为了解决这些未得到满足的需求，我们建议开发Prodige-一种用于联合 在同一单个细胞中不需要遗传的蛋白质-DNA相互作用和基因表达的图谱 操纵。它是通过可逆的细胞固定、抗体引导的染色质的战略整合而实现的 标记化，新型可变形和可降解的条形码水凝胶珠，液滴微流体，以及下一代- 世代排序技术。在目标1中，我们将开发技术组件和分析管道 Prodige。我们将制备用于抗体引导的Tn5标记的蛋白A-Tn5转座体，制作 用于单细胞产物分析的微流体装置，并合成可变形和可变形的条形码库 用于单细胞条码的可降解条码水凝胶珠。在目标2中，我们将对该方法进行批量和 单细胞水平，并评估PRODGE(联合分析)与其他单机版相比的性能 对口单位。在目标3中，我们将对Prodige进行基准测试，以建立用于联合单细胞蛋白质图谱的工具- DNA相互作用和转录组。拟议计划的成功实施将带来高度的 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的吞吐量工具 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，并确定调节细胞类型相关转录程序的蛋白质介导的机制 在不同的细胞群体中以及在不同的发育和/或疾病条件下。

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.