期刊
NATURE PROTOCOLS
卷 15, 期 3, 页码 840-876出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/s41596-019-0269-9
关键词
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资金
- European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Program [724429]
- Labex EpiGenMed, an 'Investments for the Future' program [ANR-10-LABX-12-01]
- French National Research Agency [ANR-10-INBS-04]
- European Research Council (ERC) [724429] Funding Source: European Research Council (ERC)
Here, the authors provide detailed experimental and analytical procedures for Hi-M, a method that enables simultaneous imaging of 3D genome folding and RNA localization in single cells. 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.
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