{"paper_id":"0ac00b8f-e814-45ed-9383-a18814414404","body_text":"Abstract\nThe Drosophila central brain is remarkably heterogeneous. Molecular as well as connectomic data indicate that many of its ca. 65,000 neurons are present in only a few copies per brain. This makes molecular characterization of individual cell types challenging. To address this issue, we developed Elution-based INTACT (El-INTACT), a nuclei-based purification technique that offers much higher purity, yield and efficiency compared to existing methods. El-INTACT successfully isolated two subsets of circadian neuron nuclei, one with ∼120 nuclei per brain and the other with only ∼16 nuclei per brain; the latter are the well-known circadian LNvs or PDF neurons. The method facilitated ATAC-Seq (Assay for Transposase Accessible Chromatin) assays, which revealed temporally regulated enhancers that cycle throughout the day within clock neurons. The ATAC-seq assays also identified cell-type-specific enhancers that correlate with adjacent genes, which are also only expressed in certain neuron types. To test the functional relevance of these enhancers, we developed a second approach, a CRISPR/Cas9 and multiplexed guide-RNA strategy and used it to disrupt individual enhancers of the core circadian gene Clock (Clk). Our results show that these two methods, El-INTACT to identify enhancers and a CRISPR-based strategy to perturb these enhancers, significantly improves the understanding of gene regulation within discrete neuron types.\nCompeting Interest Statement\nThe authors have declared no competing interest.","source_license":"CC-BY-4.0","license_restricted":false}