Single-cell RNA sequencing of primary mouse brown adipocytes
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ABSTRACT:
PROVIDER: PRJNA515724 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8452033.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: To test if there is a heterogeneity within brown adipocytes, we performed single cell RNA-sequencing of primary brown adipocytes isolated from mouse brown adipose tissue. Methods: Brown adipocytes from 10-week-old C57BL/6J male mice were freshly collected and resuspended in PBS containing 0.04% BSA at a concentration of 700~1200 cells/µl. Cell number and viability were measured using a TC20 Automated Cell Counter (BioRad). Single-cell RNA libraries were prepared according to the Chromium™ Single Cell 3' Reagent Kits v2 User Guide (10x Genomics). Approximately 10,000 cells were loaded on a Chromium single cell Controller instrument (10x Genomics) to generate single cell gel beads in emulsion (GEMs). The barcoded sequencing libraries were constructed using the Chromium Single-Cell 3′ Library Kit (10x Genomics) for enzymatic fragmentation, end-repair, A-tailing, adaptor ligation, ligation cleanup, sample index PCR, and PCR cleanup. Libraries were sequenced with a Hiseq 2500 instrument (Illumina) with a depth of 50k-100k reads per cell. Raw sequencing data were processed using the 10x Genomics Cell Ranger pipeline (version 2.0) to generate FASTQ files and aligned to mm10 genome to gene expression count. The subsequent data analysis was performed using “Seurat” package and R scripts. Cells with mitochondrial read rate > 50% and < 200 detectable genes were considered as low-quality and filtered out. Normalized and scaled data were clustered using the top significant principal components of highly variable genes. The t-distributed stochastic neighbor embedding (t-SNE) algorithm was used to visualize the resulting clusters. Cluster-specific markers were identified to generate heatmap and feature plots in the identified cell clusters. Genes were compared between different clusters using Bioconductor package “Limma” on normalized data. Gene Set Enrichment Analysis (GSEA) v3 was performed using genes ranked by the fold changes between different clusters to evaluate the significant activation of the C2 KEGG gene sets in MSigDb (http://software.broadinstitute.org/gsea/msigdb/collections.jsp). Results: Two major brown adipocyte subpopulations were clustered as brown adipocytes with high thermogenic activity (BA-H, 2,352 cells) and brown adipocytes with low thermogenic activity (BA-L, 1,250 cells). Conclusions: In summary, the brown adipocyte single cell transcriptomic reveals a unique subpopulation of brown adipocytes with low thermogenic activity. These cells hold a unique metabolic status, and the function of these cells is fundamentally different from the cells within the BA-H subpopulation.
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Project description:We analyzed coding transcript abundance in fully differentiated, primary brown adipocytes from murine, interscapular brown adipose tissue of wildtype and UCP1-KO mice.
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Project description:The adipose organ, including white and brown adipose tissues, is an important player in systemic energy homeostasis, storing excess energy in form of lipids while releasing energy upon various energy demands. Recent studies have demonstrated that white and brown adipocytes also function as endocrine cells and regulate systemic metabolism by secreting factors that act locally and systemically. However, a comparative proteomic analysis of secreted factors from white and brown adipocytes and their responsiveness to adrenergic stimulation has not been reported yet. Therefore, we studied and compared the secretome of white and brown adipocytes, with and without norepinephrine (NE) stimulation. Our results reveal that in the absence of NE, carbohydrate metabolism-regulating proteins are preferably secreted from white adipocytes, while brown adipocytes predominantly secrete integrin signaling proteins. Upon NE stimulation, white adipocytes secrete more proteins involved in lipid metabolism, while brown adipocytes secrete more proteins with specific anti-inflammatory properties. In conclusion, our study provides a comprehensive catalogue of novel adipokine candidates secreted from white and brown adipocytes with many of them responsive to NE.
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Project description:Brown adipocytes are specialized for heat generation and energy expenditure as a defense against cold and obesity. Recent studies demonstrate that brown adipocytes arise in vivo from a Myf5-positive, myoblastic progenitor by the action of PRDM16. Here, we identified a brown fat-enriched miRNA cluster mir-193b-365 as a key regulator of brown fat development. Blocking miR-193b and/or miR-365 in primary brown preadipocytes dramatically impaired brown adipocyte adipogenesis whereas myogenic markers were significantly induced. Forced expression of miR-193b and/or miR-365 in C2C12 myoblasts blocked the entire program of myogenesis, and miR-193b induced myoblasts to differentiate into brown adipocytes. Mir-193b-365 was upregulated by PRDM16. Our results demonstrate that mir-193b-365 serves as an essential regulator for brown fat differentiation, in part by repressing myogenesis.
2011-05-02 | GSE27614 | GEO
Project description:Brown adipocytes are specialized for heat generation and energy expenditure as a defense against cold and obesity. Recent studies demonstrate that brown adipocytes arise in vivo from a Myf5-positive, myoblastic progenitor by the action of PRDM16. Here, we identified a brown fat-enriched miRNA cluster mir-193b-365 as a key regulator of brown fat development. Blocking miR-193b and/or miR-365 in primary brown preadipocytes dramatically impaired brown adipocyte adipogenesis whereas myogenic markers were significantly induced. Forced expression of miR-193b and/or miR-365 in C2C12 myoblasts blocked the entire program of myogenesis, and miR-193b induced myoblasts to differentiate into brown adipocytes. Mir-193b-365 was upregulated by PRDM16. Our results demonstrate that mir-193b-365 serves as an essential regulator for brown fat differentiation, in part by repressing myogenesis. To study if miR-193b-365 is required for brown adipocyte adipogenesis, mRNAs from cultured primary brown adipocytes (Day 4) transfected with each locked nucleic acid (LNA) miRNA inhibitor or Control inhibitor were analyzed by microarray analysis.
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Project description:Classical brown adipocytes in interscapular BAT (Myf-5 derived) and inducible beige cells in WAT (non-Myf-5 derived) have distinct developmental origins, although both cell types have morphological and biochemical characteristics of brown fat such as the expression of UCP1. This raises an important question as to how similar the two types of brown adipocytes are at molecular and functional levels. To this end, we employed RNA-seq to systematically determine the transcriptional signatures unique to each cell type.
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| PRJEB6459 | ENA