AbstractCohesin regulates gene expression through context-specific chromatin folding mechanisms such as enhancer–promoter looping and topologically associating domain (TAD) formation by cooperating with factors such as cohesin loaders and the insulation factor CTCF. We developed a computational workflow to explore how three-dimensional (3D) structure and gene expression are regulated collectively or individually by cohesin and related factors. The main component is CustardPy, by which multi-omics datasets are compared systematically. To validate our methodology, we generated 3D genome, transcriptome, and epigenome data before and after depletion of cohesin and related factors and compared the effects of depletion. We observed diverse effects on the 3D genome and transcriptome, and gene expression changes were correlated with the splitting of TADs caused by cohesin loss. We also observed variations in long-range interactions across TADs, which correlated with their epigenomic states. These computational tools and datasets will be valuable for 3D genome and epigenome studies.