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1.
Science ; 371(6532)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33384301

RESUMO

Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms.


Assuntos
Genoma Humano , Genoma , Análise de Sequência de DNA , Animais , Sequência de Bases , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Cromatina/química , Cromatina/ultraestrutura , Posicionamento Cromossômico , Cromossomos Humanos/ultraestrutura , Cromossomos de Mamíferos/ultraestrutura , Embrião de Mamíferos , Desenvolvimento Embrionário , Epigênese Genética , Fibroblastos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Análise de Célula Única , Análise Espacial
2.
Science ; 371(6528)2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33509999

RESUMO

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.


Assuntos
Perfilação da Expressão Gênica/métodos , Imagem Molecular/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Espinhas Dendríticas , Feminino , Humanos , Camundongos , Córtex Visual
3.
Nat Methods ; 17(8): 822-832, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32719531

RESUMO

There is a need for methods that can image chromosomes with genome-wide coverage, as well as greater genomic and optical resolution. We introduce OligoFISSEQ, a suite of three methods that leverage fluorescence in situ sequencing (FISSEQ) of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how four rounds of sequencing are sufficient to produce 3D maps of 36 genomic targets across six chromosomes in hundreds to thousands of cells, implying a potential to image thousands of targets in only five to eight rounds of sequencing. We also use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunocytochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes.


Assuntos
Coloração Cromossômica/métodos , Cromossomos/química , Cromossomos/genética , Genoma Humano , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de Oligonucleotídeos , Mapeamento Físico do Cromossomo
4.
Proc Natl Acad Sci U S A ; 114(50): E10799-E10808, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29162696

RESUMO

Expansion microscopy (ExM) allows scalable imaging of preserved 3D biological specimens with nanoscale resolution on fast diffraction-limited microscopes. Here, we explore the utility of ExM in the larval and embryonic zebrafish, an important model organism for the study of neuroscience and development. Regarding neuroscience, we found that ExM enabled the tracing of fine processes of radial glia, which are not resolvable with diffraction-limited microscopy. ExM further resolved putative synaptic connections, as well as molecular differences between densely packed synapses. Finally, ExM could resolve subsynaptic protein organization, such as ring-like structures composed of glycine receptors. Regarding development, we used ExM to characterize the shapes of nuclear invaginations and channels, and to visualize cytoskeletal proteins nearby. We detected nuclear invagination channels at late prophase and telophase, potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic studies of how molecular components are configured in multiple contexts of interest to neuroscience and developmental biology.


Assuntos
Microscopia/métodos , Peixe-Zebra/anatomia & histologia , Animais , Encéfalo/ultraestrutura , Núcleo Celular/ultraestrutura , Biologia do Desenvolvimento/métodos , Larva/anatomia & histologia , Neurociências/métodos , Sinapses/ultraestrutura , Peixe-Zebra/embriologia
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