Quantification of interaction strength between a synthetic enhancer-promoter pair with Region Capture Micro-C

We mapped the 3D genomic structure of these loci across multiple clonal mouse embryonic stem cell (mESC) lines featuring varying E-P genomic distances and CTCF binding site configurations, along with control lines with either the enhancer or the entire E-P pair deleted.

Furthermore, we profiled E-P interactions under acute depletion of CTCF and/or cohesin subunit RAD21. For the 339 kb E-P pair flanked by convergent CTCF binding sites, we assessed three perturbations: CTCF depletion (via dTAG-13), RAD21 depletion (via 5-ph-IAA), and simultaneous CTCF and RAD21 co-depletion. RAD21 depletion was also performed on the 339 kb E-P pair lacking CTCF binding sites.