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1.
Nat Methods ; 18(11): 1333-1341, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34725479

RESUMO

The recent development of experimental methods for measuring chromatin state at single-cell resolution has created a need for computational tools capable of analyzing these datasets. Here we developed Signac, a comprehensive toolkit for the analysis of single-cell chromatin data. Signac enables an end-to-end analysis of single-cell chromatin data, including peak calling, quantification, quality control, dimension reduction, clustering, integration with single-cell gene expression datasets, DNA motif analysis and interactive visualization. Through its seamless compatibility with the Seurat package, Signac facilitates the analysis of diverse multimodal single-cell chromatin data, including datasets that co-assay DNA accessibility with gene expression, protein abundance and mitochondrial genotype. We demonstrate scaling of the Signac framework to analyze datasets containing over 700,000 cells.

2.
Plant Cell ; 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34791424

RESUMO

Rice (Oryza sativa) was domesticated around 10,000 years ago and has developed into a staple for half of humanity. The crop evolved and is currently grown in stably wet and intermittently dry agro-ecosystems, but patterns of adaptation to differences in water availability remain poorly understood. While previous field studies have evaluated plant developmental adaptations to water deficit, adaptive variation in functional and hydraulic components, particularly in relation to gene expression, has received less attention. Here, we take an evolutionary systems biology approach to characterize adaptive drought resistance traits across roots and shoots. We find that rice harbors heritable variation in molecular, physiological, and morphological traits that is linked to higher fitness under drought. We identify modules of co-expressed genes that are associated with adaptive drought avoidance and tolerance mechanisms. These expression modules showed evidence of polygenic adaptation in rice subgroups harboring accessions that evolved in drought-prone agro-ecosystems. Fitness-linked expression patterns allowed us to identify the drought-adaptive nature of optimizing photosynthesis and interactions with arbuscular mycorrhizal fungi. Taken together, our study provides an unprecedented, integrative view of rice adaptation to water-limited field conditions.

3.
Nature ; 597(7875): 196-205, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497388

RESUMO

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.


Assuntos
Movimento Celular , Rastreamento de Células , Células/citologia , Biologia do Desenvolvimento/métodos , Embrião de Mamíferos/citologia , Feto/citologia , Disseminação de Informação , Organogênese , Adulto , Animais , Atlas como Assunto , Técnicas de Cultura de Células , Sobrevivência Celular , Visualização de Dados , Feminino , Humanos , Imageamento Tridimensional , Masculino , Modelos Animais , Organogênese/genética , Organoides/citologia , Células-Tronco/citologia
4.
Cell ; 184(13): 3573-3587.e29, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34062119

RESUMO

The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.


Assuntos
SARS-CoV-2/imunologia , Análise de Célula Única/métodos , Células 3T3 , Animais , COVID-19/imunologia , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Humanos , Imunidade/imunologia , Leucócitos Mononucleares/imunologia , Linfócitos/imunologia , Camundongos , Análise de Sequência de RNA/métodos , Transcriptoma/imunologia , Vacinação
5.
Nat Biotechnol ; 39(10): 1246-1258, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34083792

RESUMO

Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.


Assuntos
RNA-Seq/métodos , Análise de Célula Única/métodos , Diferenciação Celular , Linhagem da Célula , Cromatina/genética , Cromatina/metabolismo , DNA Mitocondrial/genética , Epigenômica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hematopoese , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Proteínas/genética , Proteínas/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo
6.
Nat Immunol ; 22(6): 723-734, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33958784

RESUMO

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.


Assuntos
Autorrenovação Celular/imunologia , Células-Tronco Hematopoéticas/fisiologia , Reconstituição Imune , Células-Tronco Multipotentes/fisiologia , Estresse Fisiológico/imunologia , Adulto , Animais , Autorrenovação Celular/genética , Células Cultivadas , Epigênese Genética/imunologia , Feminino , Sangue Fetal/citologia , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Hematopoese , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Separação Imunomagnética , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Nectinas/metabolismo , Cultura Primária de Células , RNA-Seq , Análise de Célula Única , Sirtuína 1/metabolismo , Estresse Fisiológico/genética , Transplante Heterólogo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo
7.
Nat Genet ; 53(3): 322-331, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33649593

RESUMO

The expression of inhibitory immune checkpoint molecules, such as programmed death-ligand (PD-L)1, is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here, we apply expanded CRISPR-compatible (EC)CITE-seq, a technology that combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1 and leverage our multimodal data to identify both transcriptional and post-transcriptional modes of regulation. Specifically, we discover that the Kelch-like protein KEAP1 and the transcriptional activator NRF2 mediate the upregulation of PD-L1 after interferon (IFN)-γ stimulation. Our results identify a new mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multimodal single-cell perturbation screens.


Assuntos
Antígeno B7-H1/genética , Proteínas de Checkpoint Imunológico/fisiologia , Análise de Célula Única/métodos , Antígeno B7-2/metabolismo , Antígeno B7-H1/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas Culina/genética , Proteínas Culina/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptores de Interferon/genética , Reprodutibilidade dos Testes , Razão Sinal-Ruído , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Nat Commun ; 11(1): 5504, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127880

RESUMO

Single-cell RNA-sequencing (scRNA-Seq) is a compelling approach to directly and simultaneously measure cellular composition and state, which can otherwise only be estimated by applying deconvolution methods to bulk RNA-Seq estimates. However, it has not yet become a widely used tool in population-scale analyses, due to its prohibitively high cost. Here we show that given the same budget, the statistical power of cell-type-specific expression quantitative trait loci (eQTL) mapping can be increased through low-coverage per-cell sequencing of more samples rather than high-coverage sequencing of fewer samples. We use simulations starting from one of the largest available real single-cell RNA-Seq data from 120 individuals to also show that multiple experimental designs with different numbers of samples, cells per sample and reads per cell could have similar statistical power, and choosing an appropriate design can yield large cost savings especially when multiplexed workflows are considered. Finally, we provide a practical approach on selecting cost-effective designs for maximizing cell-type-specific eQTL power which is available in the form of a web tool.


Assuntos
Locos de Características Quantitativas/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Sequência de Bases , Biologia Computacional , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Genômica , Humanos
9.
Cell ; 182(3): 641-654.e20, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615085

RESUMO

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.


Assuntos
Diferenciação Celular/imunologia , Encefalomielite Autoimune Experimental/imunologia , Glucose-6-Fosfato Isomerase/metabolismo , Glicólise/genética , Fosforilação Oxidativa , Via de Pentose Fosfato/fisiologia , Células Th17/metabolismo , Animais , Hipóxia Celular/genética , Hipóxia Celular/imunologia , Quimera/genética , Cromatografia Gasosa , Cromatografia Líquida , Infecções por Clostridium/imunologia , Citocinas/deficiência , Citocinas/genética , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Glucose-6-Fosfato Isomerase/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Glicólise/imunologia , Homeostase/genética , Homeostase/imunologia , Inflamação/genética , Inflamação/imunologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Membrana Mucosa/imunologia , Membrana Mucosa/metabolismo , Membrana Mucosa/microbiologia , Via de Pentose Fosfato/genética , Via de Pentose Fosfato/imunologia , RNA-Seq , Análise de Célula Única , Células Th17/imunologia , Células Th17/patologia
10.
Nature ; 578(7796): 572-576, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32051590

RESUMO

Levels of gene expression underpin organismal phenotypes1,2, but the nature of selection that acts on gene expression and its role in adaptive evolution remain unknown1,2. Here we assayed gene expression in rice (Oryza sativa)3, and used phenotypic selection analysis to estimate the type and strength of selection on the levels of more than 15,000 transcripts4,5. Variation in most transcripts appears (nearly) neutral or under very weak stabilizing selection in wet paddy conditions (with median standardized selection differentials near zero), but selection is stronger under drought conditions. Overall, more transcripts are conditionally neutral (2.83%) than are antagonistically pleiotropic6 (0.04%), and transcripts that display lower levels of expression and stochastic noise7-9 and higher levels of plasticity9 are under stronger selection. Selection strength was further weakly negatively associated with levels of cis-regulation and network connectivity9. Our multivariate analysis suggests that selection acts on the expression of photosynthesis genes4,5, but that the efficacy of selection is genetically constrained under drought conditions10. Drought selected for earlier flowering11,12 and a higher expression of OsMADS18 (Os07g0605200), which encodes a MADS-box transcription factor and is a known regulator of early flowering13-marking this gene as a drought-escape gene11,12. The ability to estimate selection strengths provides insights into how selection can shape molecular traits at the core of gene action.


Assuntos
Regulação da Expressão Gênica de Plantas , Oryza/genética , Seleção Genética/genética , Secas , Evolução Molecular , Flores/genética , Flores/crescimento & desenvolvimento , Aptidão Genética/genética , Oryza/crescimento & desenvolvimento , Fotossíntese/genética , Folhas de Planta/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Tempo , Fatores de Transcrição/metabolismo
11.
Cell ; 179(7): 1455-1467, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31835027

RESUMO

Understanding the genetic and molecular drivers of phenotypic heterogeneity across individuals is central to biology. As new technologies enable fine-grained and spatially resolved molecular profiling, we need new computational approaches to integrate data from the same organ across different individuals into a consistent reference and to construct maps of molecular and cellular organization at histological and anatomical scales. Here, we review previous efforts and discuss challenges involved in establishing such a common coordinate framework, the underlying map of tissues and organs. We focus on strategies to handle anatomical variation across individuals and highlight the need for new technologies and analytical methods spanning multiple hierarchical scales of spatial resolution.


Assuntos
Variação Anatômica , Diagnóstico por Imagem/normas , Exame Físico/normas , Diagnóstico por Imagem/métodos , Humanos , Exame Físico/métodos , Padrões de Referência
12.
Genome Biol ; 20(1): 296, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870423

RESUMO

Single-cell RNA-seq (scRNA-seq) data exhibits significant cell-to-cell variation due to technical factors, including the number of molecules detected in each cell, which can confound biological heterogeneity with technical effects. To address this, we present a modeling framework for the normalization and variance stabilization of molecular count data from scRNA-seq experiments. We propose that the Pearson residuals from "regularized negative binomial regression," where cellular sequencing depth is utilized as a covariate in a generalized linear model, successfully remove the influence of technical characteristics from downstream analyses while preserving biological heterogeneity. Importantly, we show that an unconstrained negative binomial model may overfit scRNA-seq data, and overcome this by pooling information across genes with similar abundances to obtain stable parameter estimates. Our procedure omits the need for heuristic steps including pseudocount addition or log-transformation and improves common downstream analytical tasks such as variable gene selection, dimensional reduction, and differential expression. Our approach can be applied to any UMI-based scRNA-seq dataset and is freely available as part of the R package sctransform, with a direct interface to our single-cell toolkit Seurat.


Assuntos
Análise de Sequência de RNA , Análise de Célula Única , Software , Humanos , Análise de Regressão
14.
Nat Cell Biol ; 21(6): 674-686, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160712

RESUMO

In vertebrates, multipotent progenitors located in the pharyngeal mesoderm form cardiomyocytes and branchiomeric head muscles, but the dynamic gene expression programmes and mechanisms underlying cardiopharyngeal multipotency and heart versus head muscle fate choices remain elusive. Here, we used single-cell genomics in the simple chordate model Ciona to reconstruct developmental trajectories forming first and second heart lineages and pharyngeal muscle precursors and characterize the molecular underpinnings of cardiopharyngeal fate choices. We show that FGF-MAPK signalling maintains multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a pan-cardiac programme, shared by the first and second heart lineages, to define heart identity. In the second heart lineage, a Tbx1/10-Dach pathway actively suppresses the first heart lineage programme, conditioning later cell diversity in the beating heart. Finally, cross-species comparisons between Ciona and the mouse evoke the deep evolutionary origins of cardiopharyngeal networks in chordates.


Assuntos
Ciona intestinalis/genética , Coração/crescimento & desenvolvimento , Músculos Faríngeos/crescimento & desenvolvimento , Proteínas com Domínio T/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Ciona intestinalis/crescimento & desenvolvimento , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genômica , Mesoderma/crescimento & desenvolvimento , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética
15.
Cell ; 177(7): 1888-1902.e21, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31178118

RESUMO

Single-cell transcriptomics has transformed our ability to characterize cell states, but deep biological understanding requires more than a taxonomic listing of clusters. As new methods arise to measure distinct cellular modalities, a key analytical challenge is to integrate these datasets to better understand cellular identity and function. Here, we develop a strategy to "anchor" diverse datasets together, enabling us to integrate single-cell measurements not only across scRNA-seq technologies, but also across different modalities. After demonstrating improvement over existing methods for integrating scRNA-seq data, we anchor scRNA-seq experiments with scATAC-seq to explore chromatin differences in closely related interneuron subsets and project protein expression measurements onto a bone marrow atlas to characterize lymphocyte populations. Lastly, we harmonize in situ gene expression and scRNA-seq datasets, allowing transcriptome-wide imputation of spatial gene expression patterns. Our work presents a strategy for the assembly of harmonized references and transfer of information across datasets.


Assuntos
Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica , Análise de Sequência de RNA , Análise de Célula Única , Software , Transcriptoma , Humanos
16.
Nat Methods ; 16(5): 409-412, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011186

RESUMO

Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genética , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , Camundongos , Células NIH 3T3 , RNA Guia/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas
17.
Nature ; 569(7755): 222-228, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30971824

RESUMO

The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.


Assuntos
Medula Óssea/irrigação sanguínea , Microambiente Celular , Hematopoese , Células-Tronco Hematopoéticas , Análise de Célula Única , Nicho de Células-Tronco , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Linhagem da Célula , Endotélio Vascular/citologia , Feminino , Regulação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Células Mieloides/citologia , Células Mieloides/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA-Seq , Receptores Notch/metabolismo , Nicho de Células-Tronco/genética , Estresse Fisiológico/genética , Transcriptoma/genética
18.
Nat Rev Genet ; 20(5): 257-272, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30696980

RESUMO

The recent maturation of single-cell RNA sequencing (scRNA-seq) technologies has coincided with transformative new methods to profile genetic, epigenetic, spatial, proteomic and lineage information in individual cells. This provides unique opportunities, alongside computational challenges, for integrative methods that can jointly learn across multiple types of data. Integrated analysis can discover relationships across cellular modalities, learn a holistic representation of the cell state, and enable the pooling of data sets produced across individuals and technologies. In this Review, we discuss the recent advances in the collection and integration of different data types at single-cell resolution with a focus on the integration of gene expression data with other types of single-cell measurement.


Assuntos
Biologia Computacional/métodos , Mineração de Dados/estatística & dados numéricos , RNA/genética , Análise de Célula Única/estatística & dados numéricos , Conjuntos de Dados como Assunto , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas/genética , Proteínas/metabolismo , RNA/química , RNA/metabolismo , Análise de Célula Única/métodos
19.
Genome Biol ; 19(1): 224, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30567574

RESUMO

Despite rapid developments in single cell sequencing, sample-specific batch effects, detection of cell multiplets, and experimental costs remain outstanding challenges. Here, we introduce Cell Hashing, where oligo-tagged antibodies against ubiquitously expressed surface proteins uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its original sample, robustly identify cross-sample multiplets, and "super-load" commercial droplet-based systems for significant cost reduction. We validate our approach using a complementary genetic approach and demonstrate how hashing can generalize the benefits of single cell multiplexing to diverse samples and experimental designs.


Assuntos
Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Células 3T3 , Animais , Genômica , Células HEK293 , Humanos , Técnicas Imunológicas , Camundongos , Oligonucleotídeos
20.
J Exp Med ; 215(11): 2815-2832, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30291161

RESUMO

Adult hematopoiesis has been studied in terms of progenitor differentiation potentials, whereas its kinetics in vivo is poorly understood. We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSCs) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady-state. Labeled cells, comprising primarily long-term HSCs and some short-term HSCs, produced megakaryocytic lineage progeny within 1 wk in a process that required only two to three cell divisions. Erythroid and myeloid progeny emerged simultaneously by 2 wk and included a progenitor population with expression features of both lineages. Myeloid progenitors at this stage showed diversification into granulocytic, monocytic, and dendritic cell types, and rare intermediate cell states could be detected. In contrast, lymphoid differentiation was virtually absent within the first 3 wk of tracing. These results show that continuous differentiation of HSCs rapidly produces major hematopoietic lineages and cell types and reveal fundamental kinetic differences between megakaryocytic, erythroid, myeloid, and lymphoid differentiation.


Assuntos
Células-Tronco Adultas/imunologia , Diferenciação Celular/imunologia , Divisão Celular/imunologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Adultas/citologia , Animais , Células Dendríticas/citologia , Células Dendríticas/imunologia , Granulócitos/citologia , Granulócitos/imunologia , Células-Tronco Hematopoéticas/citologia , Cinética , Megacariócitos/citologia , Megacariócitos/imunologia , Camundongos , Camundongos Transgênicos , Monócitos/citologia , Monócitos/imunologia
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