Hi-M imaging of chromatin architecture in adult Drosophila brain cryosections

RESUMO

Hi-M combines fluorescence in situ hybridization (FISH), automated microfluidics, sequential imaging, and computational chromatin tracing to measure the three-dimensional organization of selected genomic regions in single cells. This chapter describes a Hi-M workflow adapted for cryosections of adult Drosophila melanogaster brains, enabling chromatin tracing while preserving tissue architecture and cell identity. The protocol covers Oligopaint library design and amplification, fixation, brain dissection, cryoprotection, cryosectioning, sequential RNA-FISH for cell-type identification, sequential DNA-FISH labeling, automated acquisition, and chromatin trace reconstruction. We also provide practical guidance for experimental design, sample preparation, tissue handling, image acquisition, and data analysis, highlighting critical steps that influence tissue integrity, hybridization efficiency, image registration, barcode detection, and chromatin trace reconstruction. The workflow is readily adaptable to different genomic loci and cell types, providing a robust approach for studying 3D genome organization in intact adult tissues.