Advancing the Biological Insights of Chromatin Accessibility Profiling: Improved Methodologies Spanning From Bulk Populations Down to Single-Cell Resolution

Abstract

Chromatin accessibility profiling is a cornerstone in epigenomic exploration, providing insights into the regulatory mechanisms that orchestrate gene expression. Understanding these mechanisms is critical as they establish the nexus between genotype and phenotype, influencing cellular identity, fate determination, and responses to environmental cues. Genome-wide methods relying on high-throughput sequencing, such as ATAC-seq (Assay for Transposase Accessible Chromatin sequencing), have emerged as powerful tools in this domain, enabling the identification of open chromatin regions where transcription factors and other regulatory proteins interact with DNA to modulate gene activity. In this dissertation, we present significant improvements in the measurement of chromatin accessibility by optimizing bulk ATAC-seq protocols for both native and fixed samples and developing a large-scale single-cell ATAC-seq method. For bulk protocol optimization, we performed a thorough analysis of 24 experimental conditions, testing all possible combinations of three reaction buffers, two enzymatic temperatures, and two enzyme sources on different nuclear preparations. This systematic evaluation, conducted on a well-characterized cell line and extended to primary mouse lung tissue, reveals intricate dependencies between protocol choices and data quality, particularly highlighting the sensitivity of chromatin accessibility data to enzymatic reaction temperatures and the physical state of nuclei. Our findings underscore the need for a multi-faceted evaluation of ATAC-seq data to mitigate protocol-driven biases and accurately represent the functional genomic elements, thereby enhancing the fidelity of chromatin profiling. Advancing beyond protocol refinement, we develop a massive-scale single-cell ATAC-seq method called txci-ATAC-seq (Ten(10)X-compatible Combinatorial Indexing ATAC Sequencing) by incorporating combinatorial indexing into a droplet-based microfluidic system. This approach substantially increases the scalability and flexibility of current single-cell assays, enabling the indexing of up to 200,000 nuclei in a single emulsion reaction across multiple samples. To improve the multiplexing capabilities of this new technique, we further develop a “phased” protocol variant (Phased-txci-ATAC-seq) that effectively decouples sample processing from library preparation, allowing for simultaneous profiling of up to 96 samples. In the proof-of-concept study, we benchmark txci-ATAC-seq across diverse biological systems, yielding an atlas of chromatin accessibility for 449,953 nuclei from different species, tissues, and genetic backgrounds. In addition, the application of txci-ATAC-seq to a CC16 knockout mouse model uncovers previously underappreciated technical artifacts derived from unintended residual genetic material introduced by gene targeting strategies, resulting in profound cell type-specific changes in chromatin landscape. The findings and methodologies established in this work expand the toolkit for epigenomic research, facilitating an in-depth examination of the intricacies of the chromatin accessibility landscape and its regulatory network.