Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M.,Nature Protocols

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Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M.
Nature Protocols ( IF 13.1 ) Pub Date : 2020-01-22 , DOI: 10.1038/s41596-019-0269-9
Andrés M Cardozo Gizzi _{1,

2} , Sergio M Espinola ₁ , Julian Gurgo ₁ , Christophe Houbron ₁ , Jean-Bernard Fiche ₁ , Diego I Cattoni ₁ , Marcelo Nollmann ₁

Affiliation

Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues.

中文翻译：

直接和同时观察Hi-M在单个细胞中的转录和染色体结构。

在单细胞水平上以高空间分辨率同时观察3D染色质的组织和转录可能是揭示调节胚胎发育，细胞分化甚至疾病的机制的关键。我们最近开发了Hi-M，该技术可在完整完整的果蝇胚胎内的单个细胞中实现多个基因组DNA基因座的顺序标记，3D成像和定位以及RNA表达。重要的是，Hi-M能够同时检测RNA表达和染色体组织，而无需卸下样品和进行初级探针杂交。在这里，我们提供了一个分步协议，描述了探针的设计，样品的制备，将胚胎稳定地固定在微流体腔室内以及完整的图像采集程序。组合的RNA / DNA荧光原位杂交过程需要4-5 d，包括收集胚胎。此外，我们描述了图像分析软件以分割核，检测基因组斑点，校正漂移并产生Hi-M矩阵。一个典型的Hi-M实验需要1-2天才能完成所有的标记和成像过程，另外需要4天来进行图像分析。这项技术可以轻松扩展，以研究培养细胞的细胞分化或复杂组织内染色质的组织。一个典型的Hi-M实验需要1-2天才能完成所有的标记和成像过程，另外需要4天来进行图像分析。这项技术可以轻松扩展，以研究培养细胞的细胞分化或复杂组织内染色质的组织。一个典型的Hi-M实验需要1-2天才能完成所有的标记和成像过程，另外需要4天来进行图像分析。这项技术可以轻松扩展，以研究培养细胞的细胞分化或复杂组织内染色质的组织。

更新日期：2020-01-22

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