Chromatin tracing at the rut locus in the Drosophila melanogaster adult brain

<ns5:p> Background The three-dimensional (3D) organization of chromatin contributes to transcriptional regulation by modulating interactions between genes and their regulatory elements. Understanding how chromatin architecture varies between neuronal cell types remains challenging, particularly in complex tissues such as the <ns5:italic>Drosophila</ns5:italic> melanogaster brain. Methods We used Hi-M, an imaging-based chromatin tracing approach, to reconstruct the 3D organization of chromatin at the rutabaga ( <ns5:italic>rut</ns5:italic> ) locus in the adult <ns5:italic>Drosophila</ns5:italic> brain. Chromatin traces were generated for the three major Kenyon cell subtypes (αβ, α′β′, and γ), for all Kenyon cells combined, and for non-Kenyon brain cells. Dataset quality was assessed through labeling efficiency measurements, comparisons between biological replicates, bootstrapping analyses, and comparison with an independent Micro-C dataset. Results We generated five cell-type-resolved chromatin tracing datasets describing chromatin organization at kilobase-scale resolution around the <ns5:italic>rut</ns5:italic> locus. All datasets showed similar labeling efficiencies, and strong reproducibility across biological replicates. Chromatin organization measured in whole brains was also in agreement with an available Micro-C dataset from the adult <ns5:italic>Drosophila</ns5:italic> central nervous system. Conclusions These datasets provide a resource for investigating cell-type-specific chromatin architecture in the adult <ns5:italic>Drosophila</ns5:italic> brain. They enable future studies of enhancer-promoter organization, chromatin folding, and the relationship between genome architecture, neuronal identity, and gene regulation. </ns5:p>