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

项目概要/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-Tn 5转座体用于抗体引导的Tn 5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。

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.