Name: isolation of adipose tissue nuclei for single-cell genomic applications
Uploaded: 2020-06-12T14:30:00
Description: Watch this Scientific Journal Video about Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications at JoVE.com

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.

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
<ol>
	<li>Prepare tissue digestion buffer.
	<ol>
		<li>Prepare ~1 mL of digestion buffer for every gram of adipose tissue.</li>
		<li>Weigh out 1.5 U/mL collagenase D and 2.4 U/mL dispase II and add phosphate buffered saline (PBS).</li>
	</ol>
	</li>
	<li>Prepare nucl...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+brown+adipose+tissue+in+healthy+adults.">Functional brown adipose tissue in healthy adults.</a> The New England Journal of Medicine. 360 (15), 1518-1525 (2009).</li><li>Nedergaard, J., Bengtsson, T., Cannon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+evidence+for+active+brown+adipose+tissue+in+adult+humans.">Unexpected evidence for active brown adipose tissue in adult humans.</a> American Journal of Physiology-Endocrinology and Metabolism. 293 (2), E444-E452 (2007).</li><li>Saito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+incidence+of+metabolically+active+brown+adipose+tissue+in+healthy+adult+humans:+effects+of+cold+exposure+and+adiposity.">High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity.</a> Diabetes. 58 (7), 1526-1531 (2009).</li><li>Yoneshiro, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruited+brown+adipose+tissue+as+an+antiobesity+agent+in+humans.">Recruited brown adipose tissue as an antiobesity agent in humans.</a> The Journal of Clinical Investigation. 123 (8), 3404-3408 (2013).</li><li>Cypess, A. 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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 &#34;cleanup&#34; step to achieve isolat...

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 &#34;browning&#34; or &#34;beiging&#34;. 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 &#946;3-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 &#34;cleanup&#34; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>autoMACS Rinsing Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>PBS with EDTA; sterile-filtered</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1595</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>21115</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell filter 100 &#956;m</td>
			<td>Corning</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell filter 40&#956;m</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics (30 &#956;m)</td>
			<td>Sysmex</td>
			<td>04-004-2326</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>P217-3</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td>Stackable filters</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>MoFloXDP Cell Sorter</td>
			<td>Beckman Coulter</td>
			<td>ML99030</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Protector RNase Inhibitor</td>
			<td>Roche</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S5-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of adipose tissue nuclei for single-cell genomic applications

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.

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 ...

Watch this Scientific Journal Video about Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications at JoVE.com

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

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 ...

Decomposition of adipose tissue into subtypes has been challenging because the fragile nature of adipocyte. This protocol was developed to overcome this by effective isolation of single nuclei. This workflow provides highly purified single nuclei with minimized nuclear aggregates and cellular debris.This is advantageous to single-cell transcriptome profiling of thousands of cells. This simple and robust protocol can be applied to study tissue-level organization of adipocyte in their adipose resident cells. And in particular, to development phenotyping, adipose knockout and transgenic mice.Visual demonstration of this protocol is critical so that people who don't have much experience in handling adipocyte can replicate and insight into adipose heterogeneity. After collecting adipose tissue from mice, pat it dry with a paper towel, and place it in a clean dish. Add calcium chloride to the digestion buffer to a final concentration of 10 mM.Then, add a small volume to the tissue and thoroughly mince it. Add about half the amount of prepared digestion buffer to the minced tissue. And use a serological pipette to transfer the sample to a 50 mL centrifuge tube.Wash the dish with the remaining buffer and add it to the 50 mL tube. Pipette the sample up and down several times. Then, incubate it for 12 to 15 minutes at 37 degrees Celsius while shaking at 200 to 210 rpm.After the incubation, add 0.5%BSA to the sample at a 1 to 1 volume ratio and mix well by pipetting. Centrifuge the sample at 300 xg for five minutes at room temperature to spin down the stromovascular fraction leaving the adipocyte fraction on top. Filter the top fraction though a 400 m cell filter into a 50 mL tube.Then, flip the filter upside down over the tube and transfer the adipocytes to a a new tube using 0.5%BSA to reverse-wash the filter. Bring the total volume of the BSA to 10 to 15 mL and pipette the sample up and down with serological pipette. Then, centrifuge it again at 300 xg for five minutes at room temperature.After centrifugation, use a wide bore pipette tip to carefully transfer the top layer of the sample to a 100 m cell filter on top of a new pre-chilled tube on ice. Rinse the filter with a sufficient amount of nuclei preparation buffer, and then discard it. From this step forward, keep a sample on ice and work quickly.Leave the sample on ice for up to two minutes intermittently inverting the tube to mix. Then, centrifuge at 1000 xg and 4 degrees Celsius for 10 minutes. Remove the supernatant and resuspend the pellet in 1 mL of nuclei preparation buffer.Transfer the sample to a clean microcentrifuge tube taking care of to avoid touching the walls of the old tube. Add 0.6 units per L RNase inhibitor and mix it. Then, centrifuge the sample for another 10 minutes.Remove the supernatant and resuspend a pellet and 1 mL of nuclei wash buffer. Double filter the sample into a clean tube using stackable 30 m filters. Then, wash the filters with about 300 L of nuclei wash buffer.To confirm successful isolation of healthy nuclei, stain a small aliquot of the sample with trypan and use an automated cell counter to assess average dead size and percent of dead cells. Stain in a small aliquot of the sample with DAPI and examine it under a microscope with a 20X objective or higher. The nuclei should be round and have an intact nuclear membrane.White arrows indicate nuclei without an intact membrane. After cleaning up the nuclei with FACS, concentrate this sample by centrifuging at 500 xg for five minutes at 4 degrees Celsius. Then, reconstitute it in an appropriate volume of nuclei wash buffer.To achieve a 50 to 1500 nuclei per L concentration and proceed with single-cell nuclei RNA seq. To remove cellular debris and doublets, adipocyte nuclei are sorted for size and granularity to exclude nuclear aggregates and multiplets. And finally for DAPI positive events.This grading strategy result in highly purified single-nuclei with minimal aggregates and debris. When inspected by microscopy, the sorted nuclei should be round and have an intact membrane. Successful purification of the nuclei can also be performed with real-time qRT-PCR using primers for nascent Ucp1 mRNA, a marker gene for brown adipocytes.The chromium platform determined a transcriptome of 7500 nuclei from murine interscapular brown adipose tissue. Seven different cell types were revealed with t-SNE dimensionality reduction and k-means clustering. All clusters expressed Fabp4, a pan adipocyte gene and a subset expressed a high level of Ucp1 mRNA.Nuclei isolated and then confirmed though this protocol can be subjected to virtually any single-cell level gene expression platform including Chromium from 10x Genomics.

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