Cell Biology of Genomes (CBGE), Center for Cancer Research (CCR), NCI/NIH, Bethesda, MD, USA.
High-Throughput Imaging Facility (HiTIF), Center for Cancer Research (CCR), NCI/NIH, Bethesda, MD, USA.
Methods Mol Biol. 2022;2532:245-274. doi: 10.1007/978-1-0716-2497-5_12.
High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.
高通量 DNA 荧光原位杂交（hiFISH）将多色组合 DNA FISH 染色与自动化图像采集和分析相结合，可在多达百万个细胞中可视化和定位数十到数百个基因组基因座。hiFISH 可用于测量基因组基因座对之间的物理距离、基因组基因座到核边缘或核中心的径向距离以及基因组基因座与蛋白质或 RNA 标记定义的核结构之间的距离。由此产生的大量三维空间距离数据集可用于研究基因组结构中的细胞异质性以及各种细胞系统中这种现象的分子机制。在本章中，我们提供了详细的 hiFISH 测量基因组基因座之间距离的方案，包括 DNA FISH 探针设计和制备、细胞培养、384 孔成像板中的 DNA FISH 染色、自动化图像采集和分析以及最终的统计分析等所有步骤。
