Development of txci-CAB as a New Single-cell Approach to Study Chromatin Modifications and Accessibility Across the Genome

Abstract

Understanding the epigenomic landscape of single cells is critical for uncovering the regulatorymechanisms that govern cell identity, differentiation, and function. Traditional chromatin profiling methods often measure a single modality per assay, limiting the ability to directly capture the interplay between chromatin accessibility and histone modifications. To overcome this limitation, we developed txci-CAB (10x-compatible combinatorial indexing of CUT&Tag and ATAC both) by combining combinatorial indexing with a droplet-based microfluidic system. In this thesis, we work on systematic optimization of ATAC-seq and CUT&Tag protocols in order to integrate them within the same workflow to simultaneously profile accessible chromatin states as well as specific modifications in individual cells. The txci-CAB protocol incorporates Tn5-based combinatorial barcoding for chromatin accessibility followed by pA-Tn5-guided CUT&Tag for histone modifications. Although several multimodal single-cell sequencing techniques exist, txci-CAB is designed to enhance scalability, sensitivity and overall convenience of use, both in terms of input reagents and protocol workflow. Our approach also emphasizes increased library complexity while maintaining robustness and reproducibility. Notably, we observed higher estimated complexity in our bulk datasets, demonstrating the method's effectiveness in simultaneously capturing two chromatin modalities at single-cell resolution.