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This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.
Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.
Studies have shown that brown adipose tissue (BAT) has a remarkable capacity to dissipate energy. Two types of thermogenic adipocytes with distinct developmental features exist in both rodents and humans: beige adipocytes and classical brown adipocytes. While classical brown adipocytes are located mostly in interscapular BAT depots, beige adipocytes sporadically emerge in white adipose tissue (WAT) in response to certain physiological cues, such as chronic cold exposure, a process referred to as "browning" or "beiging". Through the use of advanced imaging, it is now clear that adult humans have substantial depots of UCP1+ BAT, especially in the supraclavicular region1,2,3,4. The amount of adult human BAT inversely correlates with adiposity and can be increased by external cues, such as chronic cold exposure5,6 or β3-adrenergic receptor agonist7. BAT-mediated energy expenditure may offer a viable approach to combat obesity.
Until recently, thermogenic adipocytes have been considered a homogeneous population. However, studies have revealed the existence of multiple subtypes or subpopulations that are distinct in developmental origin, substrate usage, and transcriptome8,9,10. For instance, a type of beige adipocyte that preferentially uses glucose for thermogenesis, the g-beige adipocyte, was recently described10. The incomplete understanding of cell types in brown and beige adipose tissue and the lack of specific markers constitute a critical barrier to studying their biological functions.
Traditional methods for isolating subpopulations of cells are based on expression of only a few known marker genes. Recent advances in single-cell genomics enables the use of global gene expression data of single cells to provide an unbiased estimate of the number of subpopulations in a tissue. The ultimate goal of this protocol is to determine all adipose tissue subtypes under various thermogenic stimuli at a single-cell resolution. In contrast to other tissues and cell types, determining cellular subtypes of adipose tissue is challenging due to the fragility of lipid-filled adipocytes. This paper introduces a robust protocol to isolate single nuclei from adipose tissue for downstream application to snRNA sequencing. Importantly, recent literature comparing well-matched single-nuclei RNA sequencing (snRNA-seq) and single-cell RNA sequencing (scRNA-seq) datasets revealed that snRNA-seq is comparable to scRNA-seq in cell type detection, and superior in cellular coverage for a complex tissue like the brain11. This protocol combines a density gradient centrifugation method optimized for adipose tissues by Rosen et al.12 with a nuclei "cleanup" step with a MoFlo XDP High Speed Sorter. As seen in the representative results, an analysis of 7,500 single nuclei from mouse interscapular brown adipose tissue identified multiple cell types within seemingly homogeneous brown adipocytes. Overall, this simple and robust protocol can be applied to study tissue-level organization of adipocytes and adipose-resident cells, identification of subtype-specific marker genes, and development phenotyping of adipose-selective knockout/transgenic mice.
Animal care and experimentation were performed according to procedures approved by the Institutional Animal Care and Use Committee at the Albert Einstein College of Medicine.
1. Preparation of tissue digestion and lysis buffers
2. Enzymatic digestion of adipose tissue
3. Adipocyte isolation
4. Nuclei isolation
5. FACS cleanup and nuclei concentration step
Unsorted adipocyte nuclei contain debris and doublets that create noise and high background in downstream single-cell RNA sequencing. The representative FACS gate strategy is shown in Figure 1. The nuclei were first selected based on forward scatter (FSC) and side scatter (SSC) (A), then, only singlets were selected based on the combination of width and heights of SSC (B). Finally, only DAPI-positive events were selected and ...
A straightforward and robust method to isolate single nuclei and study adipose tissue heterogeneity is presented. Compared to whole tissue RNA sequencing, this workflow offers an unbiased view of cellular heterogeneity and population-specific markers. This is significant and innovative for the advancement of adipocyte biology, molecular metabolism, and obesity research.
This protocol is particularly optimized for downstream application of snRNA-seq. The "cleanup" step to achieve isolat...
The authors declare that they have no competing financial interests.
We would like to thank David Reynolds from the Albert Einstein Genomics core and Jinghang Zhang from the Flow Cytometry Core for technical support. We acknowledge support from the National Institutes of Health (NIH) (DK110426) and Pilot and Feasibility Grants from the Einstein-Mount Sinai Diabetes Research Center (DK020541), and New York Obesity Research Center (DK026687) (all to K.S.). We also would like to thank Albert Einstein Cancer Center (CA013330) for core support.
Name | Company | Catalog Number | Comments |
autoMACS Rinsing Solution | Miltenyi Biotec | 130-091-222 | PBS with EDTA; sterile-filtered |
BSA | Sigma | A1595 | |
CaCl2 | Sigma | 21115 | |
Cell filter 100 μm | Corning | 431752 | |
Cell filter 40μm | Corning | 431750 | |
CellTrics (30 μm) | Sysmex | 04-004-2326 | |
Collagenase D | Roche | 11088866001 | |
Countess II FL Automated Cell Counter | Invitrogen | AMQAF1000 | |
DAPI | Sigma | D9542 | |
Dispase II | Roche | 4942078001 | |
HEPES | Sigma | H4034 | |
KCl | Fisher | P217-3 | |
MACS SmartStrainers (30 µm) | Miltenyi Biotec | 130-098-458 | Stackable filters |
MgCl2 | Sigma | M1028 | |
MoFloXDP Cell Sorter | Beckman Coulter | ML99030 | |
NP-40 | Sigma | 74385 | |
Protector RNase Inhibitor | Roche | 3335402001 | |
Sucrose | Fisher | S5-3 |
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