| Autor: |
Liu M; Department of Genetics, Yale University School of Medicine, New Haven, CT, USA., Yang B; Department of Genetics, Yale University School of Medicine, New Haven, CT, USA., Hu M; Department of Genetics, Yale University School of Medicine, New Haven, CT, USA., Radda JSD; Department of Genetics, Yale University School of Medicine, New Haven, CT, USA., Chen Y; Department of Genetics, Yale University School of Medicine, New Haven, CT, USA., Jin S; Department of Genetics, Yale University School of Medicine, New Haven, CT, USA., Cheng Y; Department of Genetics, Yale University School of Medicine, New Haven, CT, USA., Wang S; Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. siyuan.wang@yale.edu.; Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA. siyuan.wang@yale.edu.; Yale Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA. siyuan.wang@yale.edu.; Molecular Cell Biology, Genetics and Development Program, Yale University, New Haven, CT, USA. siyuan.wang@yale.edu.; Biochemistry, Quantitative Biology, Biophysics and Structural Biology Program, Yale University, New Haven, CT, USA. siyuan.wang@yale.edu.; M.D.-Ph.D. Program, Yale University, New Haven, CT, USA. siyuan.wang@yale.edu.; Yale Center for RNA Science and Medicine, Yale University School of Medicine, New Haven, CT, USA. siyuan.wang@yale.edu.; Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA. siyuan.wang@yale.edu. |
| Abstrakt: |
The genome is hierarchically organized into several 3D architectures, including chromatin loops, domains, compartments and regions associated with nuclear lamina and nucleoli. Changes in these architectures have been associated with normal development, aging and a wide range of diseases. Despite its critical importance, understanding how the genome is spatially organized in single cells, how organization varies in different cell types in mammalian tissue and how organization affects gene expression remains a major challenge. Previous approaches have been limited by a lack of capacity to directly trace chromatin folding in 3D and to simultaneously measure genomic organization in relation to other nuclear components and gene expression in the same single cells. We have developed an image-based 3D genomics technique termed 'chromatin tracing', which enables direct 3D tracing of chromatin folding along individual chromosomes in single cells. More recently, we also developed multiplexed imaging of nucleome architectures (MINA), which enables simultaneous measurements of multiscale chromatin folding, associations of genomic regions with nuclear lamina and nucleoli and copy numbers of numerous RNA species in the same single cells in mammalian tissue. Here, we provide detailed protocols for chromatin tracing in cell lines and MINA in mammalian tissue, which take 3-4 d for experimental work and 2-3 d for data analysis. We expect these developments to be broadly applicable and to affect many lines of research on 3D genomics by depicting multiscale genomic architectures associated with gene expression, in different types of cells and tissue undergoing different biological processes. |
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