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      Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M

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          Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes.

          A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide- and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.
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            Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow.

            A lack of techniques to image multiple genomic loci in living cells has limited our ability to investigate chromosome dynamics. Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins. We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.
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              Visualizing DNA folding and RNA in embryos at single-cell resolution

              Establishment of cell types during development requires precise interactions between genes and distal regulatory sequences. Our understanding of how these interactions look in three dimensions, vary across cell types in complex tissue, and relate to transcription remains limited. Here we describe optical reconstruction of chromatin architecture (ORCA), a method to trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching 2 kilobases. We applied ORCA to a Hox gene cluster in cryosectioned Drosophila embryos and labelled ~30 RNA species in parallel. We identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and unexpected Polycomb-independent borders. Deletion of Polycomb-independent borders led to ectopic enhancer-promoter contacts, aberrant gene expression, and developmental defects. Together, these results illustrate an approach for high-resolution, single-cell DNA domain analysis in vivo, reveal domain structures that change with cell identity, and show that border elements contribute to formation of physical domains in Drosophila.
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                Author and article information

                Journal
                Nature Protocols
                Nat Protoc
                Springer Science and Business Media LLC
                1754-2189
                1750-2799
                January 22 2020
                Article
                10.1038/s41596-019-0269-9
                31969721
                2bcfee21-014c-4caf-994b-37e2f169e0bd
                © 2020

                http://www.springer.com/tdm

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