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To image the accessible genome at nanometer scale in situ , we developed 3D ATAC-PALM which integrates Assay for Transposase-Accessible Chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with Oligopaint DNA-FISH, RNA-FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially-segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

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