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Summary: Transposase-accessible chromatin by sequencing (ATAC-seq) has emerged as an advantageous technique to assess chromatin accessibility owing to the robustness of ''tagmentation'' process and a relatively faster library preparation. A comprehensive ATAC-seq protocol from Drosophila brain tissue is currently unavailable. Here, we have provided a detailed protocol of ATAC-seq assay from Drosophila brain tissue. Starting from dissection and transposition to amplification of libraries has been elaborated. Furthermore, a robust ATAC-seq analysis pipeline has been presented. The protocol can be easily adapted for other soft tissues. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.