Name: isolation of adipose tissue nuclei for single-cell genomic applications
Uploaded: 2020-06-12T14:30:00.000+00:00
Duration: 7 min 3 s
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 nuclei preparation buffer (NPB).
	<ol>
		<li>Prepare 10 mM HEPES, 1.5 mM magnesium chloride, 10 mM potassium chloride, 250 mM sucrose, and 0.1% NP-40 in nuclease-free water. Mix well. Sucrose may take more time to dissolve than the other components.</li>
	</ol>
	</li>
	<li>Prepare 0.5% bovine serum albumin (BSA) solution.
	<ol>
		<li>Prepare 0.5% BSA in PBS containing EDTA. It is recommended to use sterile-filtered cell culture grade PBS (see Table of Materials).</li>
	</ol>
	</li>
</ol>
2. Enzymatic digestion of adipose tissue
<ol>
	<li>Dissect mice to extract adipose tissue according to Aune et al.13. Collect isolated tissue in a dish containing PBS. Transfer the tissue to a paper towel to pat dry. Then place the tissue in a clean dish.</li>
	<li>Add calcium chloride to the digestion buffer to a final concentration of 10 mM. Add a small volume of digestion buffer to the dish and thoroughly mince the adipose tissue. The volume of digestion buffer required will be based on the amount of tissue isolated. Usually ~1 mL or less is used.</li>
	<li>Add about half of the amount of prepared digestion buffer (e.g., ~10 mL for five mice) to the minced tissue. Using a serological pipette, transfer the sample to a 50 mL conical centrifuge tube. Add any remaining amount of buffer to wash the dish and transfer to the 50 mL tube containing the sample. Pipette up and down several times. Then incubate with shaking at 200-210 rpm at 37 &#176;C for 12-15 min.</li>
</ol>
3. Adipocyte isolation
<ol>
	<li>After incubation, add 0.5% BSA at a 1:1 ratio to the sample. Mix well by pipetting up and down several times. Centrifuge the sample at 300 x g for 5 min at room temperature (RT) to spin down the stromal vascular fraction (SVF). The adipocyte fraction will remain in the top layer of the sample.</li>
	<li>Place a 40 &#181;m cell filter on top of a 50 mL conical centrifuge tube and filter the sample, collecting the top layer and leaving behind the SVF at the bottom of the tube. Transfer the top layer (adipocyte fraction) to a new tube by flipping the filter upside down over the tube and using 0.5% BSA to reverse wash the filter and collect the sample in the tube.</li>
	<li>Bring the total volume of 0.5% BSA to 10-15 mL based on sample size and pipette up and down with a serological pipette or briefly shake up and down to mix the sample. Centrifuge the sample again at 300 x g for 5 min at RT.</li>
</ol>
4. Nuclei isolation
<ol>
	<li>Use a wide-bore pipette tip and carefully transfer the top layer of the sample to a 100 &#181;m cell filter placed on top of a new prechilled tube on ice, leaving behind any residual SVF.</li>
	<li>Rinse/wash the filter with a sufficient amount of NPB and discard the filter. From this step forward have the sample on ice as often as possible and work quickly to avoid leaky and/or unhealthy nuclei. It is helpful to prechill microcentrifuge tubes to be used in future steps on ice.</li>
	<li>Place the sample on ice for no more than 2 min with intermittent gentle inverting of the tube to mix. The incubation time on ice should be optimized for sample size and type of adipocytes. Centrifuge at 1,000 x g at 4 &#176;C for 10 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 1 mL of NPB. Transfer the sample to a clean microcentrifuge tube. While transferring, avoid touching the walls of the tube, which contain debris and lipid. Add 0.6 U/&#181;L RNase inhibitor to the sample and mix. Centrifuge again at 1,000 x g at 4 &#176;C for 10 min. 
	NOTE: Centrifugation speed and time should be decreased at this step for smaller samples.</li>
	<li>Remove the supernatant and resuspend in 1 mL of Nuclei Wash Buffer (2% BSA/PBS + 0.6 U/&#181;L RNase inhibitor).</li>
	<li>Double filter into a clean tube with stackable 30 &#181;m filters. Wash filter with a small volume (~300 &#181;L) of Nuclei Wash Buffer. Alternatively, for smaller samples, use a 30 &#181;m filter that fits into a microcentrifuge tube and filter directly into it.</li>
	<li>Confirm successful isolation of healthy nuclei.
	<ol>
		<li>Stain a small aliquot of the sample with trypan and use an automated cell counter to assess average dead size and percent dead cells. Average dead size for a sample containing mostly nuclei should range between 7-10 &#181;m. Nuclei size is usually around 8 &#181;m.</li>
		<li>Stain a small aliquot of the sample with DAPI and examine under a microscope with a 20x objective or higher. The nuclear membrane should be intact and the nuclei should be round.</li>
	</ol>
	</li>
</ol>
5. FACS cleanup and nuclei concentration step
<ol>
	<li>Proceed immediately to the FACS step to clean up nuclei and remove any excess debris. The collection buffer should contain Nuclei Wash Buffer. During FACS the buffer is diluted. Preparing a higher concentration of BSA and RNase inhibitor to account for dilution during sorting is helpful. The FACS collection block should be on cooling mode.</li>
	<li>After sorting the nuclei, centrifuge at 500 x g for 5 min at 4 &#176;C to concentrate the sample. Reconstitute in an appropriate volume of Nuclei Wash Buffer to achieve a concentration of 500-1,500 nuclei/&#181;L. Do not attempt to remove all the supernatant. Doing so will result in loss of nuclei.</li>
	<li>Use a hemocytometer and a DAPI stained aliquot for a final count and to visualize the nuclei. Then proceed immediately with snRNA-seq according to the manufacturer&#39;s protocol.</li>
</ol>

<ol>
	<li>Cypess, A. 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=Identification+and+importance+of+brown+adipose+tissue+in+adult+humans.">Identification and importance of brown adipose tissue in adult humans.</a> The New England Journal of Medicine. 360 (15), 1509-1517 (2009).</li><li>van Marken Lichtenbelt, W. D., 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=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> The New England Journal of Medicine. 360 (15), 1500-1508 (2009).</li><li>Virtanen, K. 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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E., 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=Single-nucleus+and+single-cell+transcriptomes+compared+in+matched+cortical+cell+types.">Single-nucleus and single-cell transcriptomes compared in matched cortical cell types.</a> PloS One. 13 (12), e0209648 (2018).</li><li>Roh, H. C., 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=Simultaneous+Transcriptional+and+Epigenomic+Profiling+from+Specific+Cell+Types+within+Heterogeneous+Tissues+In+Vivo.">Simultaneous Transcriptional and Epigenomic Profiling from Specific Cell Types within Heterogeneous Tissues In Vivo.</a> Cell Reports. 18 (4), 1048-1061 (2017).</li><li>Aune, U. L., Ruiz, L., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+differentiation+of+stromal+vascular+cells+to+beige/brite+cells.">Isolation and differentiation of stromal vascular cells to beige/brite cells.</a> Journal of Visualized Experiments. (73), e50191 (2013).</li><li>Zheng, G. X. Y., 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=Massively+parallel+digital+transcriptional+profiling+of+single+cells.">Massively parallel digital transcriptional profiling of single cells.</a> Nature Communications. 8, 14049 (2017).</li><li>Pollen, A. 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=Low-coverage+single-cell+mRNA+sequencing+reveals+cellular+heterogeneity+and+activated+signaling+pathways+in+developing+cerebral+cortex.">Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.</a> Nature Biotechnology. 32 (10), 1053-1058 (2014).</li><li>Hayashi, 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=Single-cell+full-length+total+RNA+sequencing+uncovers+dynamics+of+recursive+splicing+and+enhancer+RNAs.">Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.</a> Nature Communications. 9 (1), 619 (2018).</li><li>Satpathy, A. 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=Massively+parallel+single-cell+chromatin+landscapes+of+human+immune+cell+development+and+intratumoral+T+cell+exhaustion.">Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion.</a> Nature Biotechnology. 37 (8), 925-936 (2019).</li><li>Stoeckius, 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=Simultaneous+epitope+and+transcriptome+measurement+in+single+cells.">Simultaneous epitope and transcriptome measurement in single cells.</a> Nature Methods. 14 (9), 865-868 (2017).</li><li>Peterson, V. 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=Multiplexed+quantification+of+proteins+and+transcripts+in+single+cells.">Multiplexed quantification of proteins and transcripts in single cells.</a> Nature Biotechnology. 35 (10), 936-939 (2017).</li><li>Gaublomme, J. 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=Nuclei+multiplexing+with+barcoded+antibodies+for+single-nucleus+genomics.">Nuclei multiplexing with barcoded antibodies for single-nucleus genomics.</a> Nature Communications. 10 (1), 2907 (2019).</li></ol>

The authors declare that they have no competing financial interests.

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 isolation of healthy nuclei with the MoFlo XDP High Speed Sorter completely removes debris and aggregates from the crude suspension while maintaining nuclear membrane integrity. Therefore, this increases capture efficiency in the droplet formation and recovers thousands of single-nuclei transcriptomes from just one run without loss of the number of detected genes. In addition to droplet-based single-cell platforms, which provide a higher number of surveyed single nuclei or cells, sorter-based microplate and Fluidigm C1 platforms15 can also be used to obtain greater resolution and coverage of the transcriptome16. Because this workflow offers high-quality nuclei, assays for transposase-accessible chromatin using sequencing (i.e., ATAC-sequencing) can also be performed with minimum modifications to the existing scATAC-seq protocol17.
One limitation of this protocol is the loss of information from the removal of nonnuclear compartments, such as the cytosol and cellular membrane. For instance, emerging multimodal single-cell analyses18,19 that simultaneously measure cell membrane protein expression and the transcriptome cannot be applied to this protocol.
The future goal is to combine this protocol with nuclei multiplexing using barcoded antibodies20. Sorted nuclei from different adipose tissues under different experimental conditions will be barcoded and pooled using an antinuclear pore complex antibody and snRNA-seq will be performed. By sequencing these barcodes alongside the nuclear transcriptome, each nuclei can be assigned to its original sample, cross-sample multiplets can be clearly identified, and droplet-based systems can be &#34;super-loaded&#34; for significant cost reduction per nuclei.

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><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 &amp;mu;m</td><td>Corning</td><td>431752</td><td></td></tr><tr><td>Cell filter 40&amp;mu;m</td><td>Corning</td><td>431750</td><td></td></tr><tr><td>CellTrics (30 &amp;mu;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 &amp;micro;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 sorted into collection buffer (C). This workflow provides highly-purified single nuclei with minimized nuclear aggregates and cellular debris (Figure 2).
To confirm the integrity of the sorted nuclei, a small aliquot of the sample was inspected by microscope after DAPI staining. The nuclear membrane should be intact and the nuclei should be round (Figure 2B). To confirm the success of the purification, it is also recommended to collect supernatant (i.e., collection buffer) after centrifugation and run real-time qRT-PCR of a marker gene. Use of a primer set (primer sequences in Table 1) designed to amplify nascent, intron-containing UCP1, confirmed that the supernatant of sorted nuclei did not contain a detectable level of nascent Ucp1 mRNA (Figure 3). This step can be done for other marker genes highly abundant in brown, beige, or white adipocytes, such as Cidea, Pgc1-a, Adipoq, or Fabp4.
Nuclei isolated and confirmed through this protocol can be subjected to virtually any single-cell level gene expression platform. Use of the Chromium platform14 (10x Genomics) determined a transcriptome of 7,500 nuclei from interscapular brown adipose tissue from 8-week-old male C57BL/6 mice (Figure 4). t-distributed stochastic neighbor embedding (t-SNE) dimensionality reduction and K-means clustering revealed seven cell types, including brown adipocytes, endothelial cells, mural cells marked by Pdgfrb, adipocyte progenitors, and immune cells. All clusters expressed Fabp4, a pan-adipocyte gene, to varying degrees (Figure 4B). A subset of clusters expressed a high level of Ucp1 mRNA, whereas other clusters sporadically expressed Ucp1 (Figure 4A,C). This is consistent with a previous study showing that interscapular brown adipose tissue contains at least two distinct populations with high and low Ucp1 expression and thermogenic activity8,9.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61230/61230fig1.jpg" /> 
Figure 1: Representative flow cytometry analysis of nuclei by MoFlo XDP High Speed Sorter. (A) Size and granularity. (B) Detection of nuclear aggregates and multiplets. (C) Final sorting gate selecting only DAPI-positive events. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61230/61230fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61230/61230fig2.jpg" /> 
Figure 2: Representative image of nuclei isolated from brown adipocytes. (A) Before and (B) after MoFLo XDP sorting. White arrows in A indicate nuclei without an intact membrane. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61230/61230fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61230/61230fig3.jpg" /> 
Figure 3: Representative qRT-PCR result for isolated nuclei and the supernatant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61230/61230fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61230/61230fig4.jpg" /> 
Figure 4: Representative 10x chromium snRNA-seq results for brown adipocytes isolated from 8-week-old male C57BL/6 mice under RT. (A) t-SNE dimensionality reduction and K-Means clustering for 7,500 nuclei. k = 9. Canonical markers used for cluster annotations are shown in parentheses. (B) mRNA expression of Fabp4, an adipocyte-selective marker, in A. White = no expression, red = high expression. (C) mRNA expression of Ucp1, thermogenic marker in A. These data are from a single experiment. snRNA-seq data used for these figures was deposited in the Gene Expression Omnibus (GEO) under superseries accession number GSE144720. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61230/61230fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Gene</td>
			<td>Species</td>
			<td>Forward primer</td>
			<td>Reverse primer</td>
		</tr>
		<tr>
			<td>Ucp1 (Intron-containing)</td>
			<td>mouse</td>
			<td>GAT CTT CTC AGC CGG AGT TTC</td>
			<td>CCT TCC TAA TAG CAC CCA TTC C</td>
		</tr>
	</tbody>
</table>
Table 1: Primer sequences for real-time qPCR analysis.

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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 ...

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9.8K Views.  Albert Einstein College of Medicine. Afbraak van vetweefsel in subtypes is een uitdaging geweest omdat de fragiele aard van adipocyte. Dit protocol werd ontwikkeld om dit te overwinnen door effectieve isolatie van enkele kernen. Deze workflow biedt zeer gezuiverde enkele kernen met geminimaliseerde nucleaire aggregaten en cellulair puin.Dit is voordelig voor single-cell transcriptome profilering van duizenden cellen. Dit eenvoudige en robuuste protocol kan worden toegepast om weefsel-niveau organisatie van adipocyte in hu...

Video: Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin accessibility profiling. This technique has been useful for understanding the regulatory mechanisms of gene expression in a range of biological processes. Although ATAC-seq has been modified for different types of samples, there have not been effective modifications of ATAC-seq methods for adipose tissues. Challenges with adipose tissues include the complex cellular heterogeneity, large lipid content, and high mitochondrial contamination. To overcome these problems, we have developed a protocol that allows adipocyte-specific ATAC-seq by employing fluorescence-activated nucleus sorting with adipose tissues from the transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mouse. This protocol produces high-quality data with minimal wasted sequencing reads while reducing the amount of nucleus input and reagents. This paper provides detailed step-by-step instructions for the ATAC-seq method validated for the use of adipocyte nuclei isolated from mouse adipose tissues. This protocol will aid in the investigation of chromatin dynamics in adipocytes upon diverse biological stimulations, which will allow for novel biological insights.

We present a protocol for assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) specifically on adipocytes using nucleus sorting with adipose tissues isolated from transgenic reporter mice with nuclear fluorescence labeling.

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Medicine

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Developmental Biology

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Immunology and Infection

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Biology

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

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