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

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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.