发表机构
Centre de Biologie Structurale, Univ Montpellier, CNRS UMR5048, INSERM U1054（结构生物学中心，蒙彼利埃大学，法国国家科学研究中心UMR5048，INSERM U1054）
机构由 AI 辅助整理，请以论文原文为准。AI 中文总结
本文介绍了一种适用于成年果蝇脑冷冻切片的Hi-M成像工作流程，结合FISH、微流控和计算追踪，在保留组织结构的同时实现染色质三维结构分析。
AI 中文摘要
Hi-M结合了荧光原位杂交（FISH）、自动化微流控、顺序成像和计算染色质追踪，以测量单细胞中选定基因组区域的三维组织。本章描述了一种适用于成年黑腹果蝇脑冷冻切片的Hi-M工作流程，使得在保留组织结构和细胞身份的同时进行染色质追踪成为可能。该方案涵盖了Oligopaint文库的设计与扩增、固定、脑解剖、冷冻保护、冷冻切片、用于细胞类型鉴定的顺序RNA-FISH、顺序DNA-FISH标记、自动化采集以及染色质轨迹重建。我们还为实验设计、样本制备、组织处理、图像采集和数据分析提供了实用指导，强调了影响组织完整性、杂交效率、图像配准、条形码检测和染色质轨迹重建的关键步骤。该工作流程可轻松适应不同的基因组位点和细胞类型，为研究完整成体组织中3D基因组组织提供了一种稳健的方法。
英文摘要
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.
