This work was supported by the IUSM Showalter Research Trust Fund (to H.C.R.), an IUSM Center for Diabetes and Metabolic Diseases Pilot and Feasibility grant (to H.C.R.), the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK129289 to H.C.R.), and the American Diabetes Association Junior Faculty Award (7-21-JDF-056 to H.C.R.).

Animal care and experimentation were performed according to procedures approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine.
1. Preparations before beginning the experiment
<ol>
	<li>Tissue preparation
	<ol>
		<li>For adipocyte nucleus labeling, cross NuTRAP mice with adipocyte-specific adiponectin-Cre lines (Adipoq-Cre) to generate Adipoq-NuTRAP mice, which are hemizygous for both Adipoq-Cre and NuTRAP.</li>
		<li>Dissect the adipose tissues of interest from the Adipoq-NuTRAP mice as described previously14.</li>
		<li>Snap-freeze the tissues in liquid nitrogen, and store them at &#8722;80 &#176;C until use. 
		NOTE: Each fat pad can be stored as a whole, and the frozen tissues can be later cut on dry ice into smaller pieces (~50 mg) for experiments. Alternatively, the fat tissues can be cut, aliquoted, and frozen in tubes for storage (~50 mg per tube). Frozen samples are never allowed to thaw after freezing until use.</li>
	</ol>
	</li>
	<li>Buffer preparation 
	NOTE: Keep the buffers on ice throughout the experiment.
	<ol>
		<li>Prepare nucleus preparation buffer (NPB): 250 mM sucrose, 10 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, and 0.1% NP40. Prepare 7 mL of NPB per sample. Add fresh 100x protease inhibitor cocktail (final 1x), DTT (final 1 mM), and Hoechst (final 1 &#181;g/mL) to the NPB before use. 
		NOTE: It is recommended to prepare fresh NPB, but it can be stored at 4 &#176;C for up to 1 month.</li>
		<li>Prepare 1x phosphate-buffered saline with 0.1% NP-40 (PBS-N).</li>
	</ol>
	</li>
</ol>
2. Nucleus isolation
<ol>
	<li>Chill glass Dounce homogenizers, with one glass Dounce per sample, on ice. Then, add 7 mL of the NPB mix to each glass Dounce. 
	NOTE: It is recommended to use up to eight samples in each experiment. Working with more than eight samples would significantly delay all the steps, and it may especially downgrade the nucleus quality during sorting.</li>
	<li>Put the frozen adipose tissue (50-100 mg) in the glass Dounce, and immediately cut it into a few smaller pieces using a long pair of scissors. Stroke 10x with a loose pestle for all the samples, and then 10x with a tight pestle. 
	NOTE: Adipose tissues are soft, so cutting them into a few small pieces should be sufficient. For rare samples, smaller pieces (10-20 mg) can be used, although the collected adipocyte nucleus number may vary.</li>
	<li>Filter through a 100 &#181;m strainer into a new 50 mL tube. Move the filtered homogenates to a new 15 mL tube by pouring. Spin at 200 &#215; g for 10 min at 4 &#176;C in a swing-bucket centrifuge. Remove the supernatant as much as possible. 
	NOTE: Aspirate first using a vacuum, and then remove the remaining volume by using a micropipette.</li>
	<li>Resuspend the pellet in 500 &#181;L of PBS-N by gentle but thorough finger tapping. 
	NOTE: Avoid pipetting, as this may damage the nuclei.</li>
	<li>Filter through a 40 &#181;m strainer into a new 50 mL tube using gentle pipetting. Rinse the strainer with 250 &#181;L of PBS-N. Transfer the filtered nuclear resuspension to a fluorescence-activated cell sorting (FACS) tube using a micropipette. 
	&#8203;NOTE: If available, tubes and cell strainers can be used for smaller volumes to minimize sample loss.</li>
</ol>
3. Nucleus sorting
<ol>
	<li>Collection tube preparation
	<ol>
		<li>Add 500 &#181;L of PBS-N to new 1.5 mL tubes, and invert a few times to wet all inner surfaces with the buffer.</li>
		<li>Briefly spin the tubes to bring down all the liquid, and then chill them on ice until sorting.</li>
	</ol>
	</li>
	<li>FACS (Figure 2)
	<ol>
		<li>Use the FSC-A/FSC-H gating for singlets and FSC-A/SSC-A for small or large debris removal.</li>
		<li>Gate for the mCherry/GFP-positive adipocyte nucleus population.</li>
		<li>Collect 10,000 nuclei in each of the collection tubes prepared in step 3.1.</li>
	</ol>
	</li>
	<li>Post-sorting nucleus preparation
	<ol>
		<li>Add 500 &#181;L of PBS-N to each collection tube, and invert a few times to mix.</li>
		<li>Spin the collection tubes at 200 &#215; g for 10 min at 4 &#176;C in a swing-bucket centrifuge. Completely remove the supernatant. 
		NOTE: Use a vacuum first to remove bubbles, pour off the supernatant, and use paper wipes to remove the remaining liquid completely. The pellet is invisible at this point. Prepare the Tn5 master mixture during the centrifugation step as described below.</li>
	</ol>
	</li>
</ol>
4. Tn5 Tagmentation (Table 1)
<ol>
	<li>To prepare 25 &#181;L of Tn5 master mixture, mix 12.5 &#181;L of 2x tagmentation DNA buffer (TD buffer), 1.25 &#181;L of Tn5 transposase (TDE I enzyme), and 11.25 &#181;L of nuclease-free water.</li>
	<li>Add 25 &#181;L of Tn5 master mixture to each nucleus pellet, and resuspend by gentle pipetting. Incubate at 600 rpm for 30 min at 37 &#176;C using a thermomixer.</li>
</ol>
5. DNA purification
NOTE: The following procedure uses the PCR purification kit mentioned in the Table of Materials. Any other similar DNA purification methods can be used.
<ol>
	<li>Add 25 &#181;L of nuclease-free water to the 25 &#181;L of the mixture of Tn5 nucleus resuspension obtained after step 4.2. The final volume is 50 &#181;L per sample.</li>
	<li>Add 250 &#181;L of buffer PB.</li>
	<li>Add 5 &#181;L of 3 M sodium acetate (NaOAc) (pH 5.2).</li>
	<li>Transfer the mixture to a column (provided with the referenced kit), and centrifuge at 17,900 &#215; g for 1 min at room temperature (RT).</li>
	<li>Discard the flowthrough, and place the spin column back in the same collection tube. Add 750 &#181;L of buffer PE containing absolute ethanol (EtOH), and centrifuge at 17,900 &#215; g for 1 min at RT.</li>
	<li>Discard the flowthrough, and place the spin column back into the same collection tube. Centrifuge at 17,900 &#215; g for 1 min at RT, and discard the collection tube with the flowthrough.</li>
	<li>To elute the DNA fragments, equip the column with a new 1.5 mL tube, add 10 &#181;L of buffer EB, and leave at RT for 3 min. Centrifuge at 17,900 &#215; g for 1 min at RT. 
	&#8203;NOTE: The samples can be stored at 4 &#176;C for days or at &#8722;20 &#176;C for weeks.</li>
</ol>
6. PCR amplification (Table 2)
NOTE: The primers used in this study are listed in Table 3.
<ol>
	<li>To amplify transposed DNA fragments, prepare PCR master mixture without adding a unique Ad2.n barcoding primer (primer #2). Use 38 &#181;L of the PCR master mixture per sample: 11 &#181;L of nuclease-free water, 2 &#181;L of 25 &#181;M Ad1_noMX primer (primer #1), and 25 &#181;L of 2x PCR Master Mix.</li>
	<li>Add 38 &#181;L of the PCR master mixture into a 0.2 mL PCR tube.</li>
	<li>Add 10 &#181;L of the eluted DNA fragments from step 5.7.</li>
	<li>Add 2 &#181;L of 25 &#181;M primer #2, which needs to be specific for each sample.</li>
	<li>Run the PCR according to the cycling conditions indicated in Table 2.</li>
</ol>
7. Real-time quantitative PCR (qPCR) test (Table 4)
NOTE: This step aims to determine the additional cycles needed to amplify the DNA. It is optional but highly recommended, especially for new experiments.
<ol>
	<li>Prepare qPCR master mixture without adding primer #2. Use 9.975 &#181;L of the qPCR master mixture per sample: 4.41 &#181;L of nuclease-free water, 0.25 &#181;L of 25 &#181;M primer #1, 5 &#181;L of 2x PCR Master Mix, and 0.09 &#181;L 100x SYBR Green I. 
	NOTE: To prepare 100x SYBR Green I, dilute the 10,000x stock with nuclease-free water. As the volume of 100x SYBR Green I used for the qPCR master mixture is negligible, it is easier to make an additional master mixture of diluted 100x SYBR Green I (e.g., for 8-10 samples).</li>
	<li>Add 9.975 &#181;L of the qPCR master mixture into each well of a qPCR plate.</li>
	<li>Add 0.25 &#181;L of 25 &#181;M primer #2 into each well. 
	NOTE: Be sure to add the same primer #2 to match what was added to each specific sample in step 6.4.</li>
	<li>Add 5 &#181;L of the PCR reaction obtained after the PCR amplification in step 6.5, and mix by pipetting. 
	NOTE: The remaining 45 &#181;L of the PCR reaction will be used for the second PCR amplification.</li>
	<li>Run the qPCR according to the cycling conditions indicated in Table 5.</li>
	<li>Check the amplification curves, and identify the number of additional PCR cycles needed by estimating the number of cycles that reached ~35% of the maximum (Figure 3A). 
	NOTE: Typically, 10,000 nuclei require five to eight additional PCR cycles.</li>
</ol>
8. Additional PCR amplification
<ol>
	<li>Run the PCR using all of the remaining 45 &#181;L of the PCR reaction according to the calculations from step 7.6 (Table 6). 
	&#8203;NOTE: Each sample could require a different number of amplification cycles.</li>
</ol>
9. Second DNA purification using the PCR purification kit
<ol>
	<li>Use the same procedures as in section 5, but elute the amplified DNA fragments with 20 &#181;L of buffer EB.</li>
</ol>
10. DNA fragment size selection
NOTE: Use solid phase reversible immobilization beads, as described below. Any other similar DNA purification beads can be used according to the manufacturer&#39;s instructions. The SPRI bead suspension should be completely resuspended and equilibrated at RT with rotation before use.
<ol>
	<li>Transfer the eluted DNA to a new PCR tube, and add 80 &#181;L of buffer EB to make a final volume of 100 &#181;L.</li>
	<li>Add 55 &#181;L of SPRI beads (0.55x sample volume), and mix by pipetting. Incubate for 5 min at RT, and then separate on a mini magnetic stand for PCR 8-tube strips for 5 min.</li>
	<li>Transfer 150 &#181;L of the supernatant to a new PCR tube. Add 95 &#181;L of SPRI beads, and mix by pipetting. 
	NOTE: The supernatant contains DNA of approximately 500 bp or smaller.</li>
	<li>Incubate for 5 min at RT, and then separate on the mini magnetic stand for 5 min. Discard the supernatant carefully.</li>
	<li>Wash the beads for 1 min with 200 &#181;L of 70% EtOH. Repeat twice for three washes in total. 
	NOTE: Do not move PCR tubes from the magnetic stand during the washes.</li>
	<li>After the final wash, spin down the tubes at 1,000 &#215; g for 1 min, and pipet out the remaining EtOH. Dry the pellet with the lid open at 37 &#176;C for 2 min on a PCR machine. 
	NOTE: The bead pellets should display only some minor cracks once dried. Avoid over-drying.</li>
	<li>Resuspend the pellet with 20 &#181;L of buffer EB by pipetting, and incubate for 5 min at RT. Separate on the mini magnetic stand for 5 min, and then transfer 18 &#181;L of the supernatant containing the final library to a new 1.5 mL tube. 
	&#8203;NOTE The library can be stored at &#8722;20 &#176;C while performing a quality check.</li>
</ol>
11. DNA quantification using a fluorometer
<ol>
	<li>To prepare the master mixture, dilute 200x dsDNA High Sensitivity (HS) reagent with dsDNA HS buffer to a final concentration of 1x.</li>
	<li>For standards, add 190 &#181;L of the 1x master mixture and 10 &#181;L of standard 1 or standard 2 to a fluorometer tube. 
	NOTE: Equilibrate the standards at RT in the dark before starting the quantification.</li>
	<li>For the library samples, add 198 &#181;L of the 1x master mixture and 2 &#181;L of each sample to a separate fluorometer tube. 
	NOTE: If the sample amounts are limited, the sample volume can be reduced to 1 &#181;L, and the 1x master mixture volume can be increased to 199 &#181;L.</li>
	<li>Vortex or shake the fluorometer tubes, and let them sit for 5 min at RT in the dark. Measure the DNA concentration using the dsDNA HS assay of the fluorometer.</li>
</ol>
12. Library quality check by high-sensitivity electrophoresis systems
<ol>
	<li>Take the ATAC-seq library, and dilute with nuclease-free water to a final concentration of 1-5 ng/&#181;L.</li>
	<li>Analyze the size distribution of the ATAC-seq libraries using high-sensitivity, automated electrophoresis systems following the manufacturer&#39;s protocols.</li>
</ol>
13. Quality check of the ATAC-seq library by using targeted qPCR
<ol>
	<li>Take 5-10 ng of the ATAC-seq library, and dilute with 50 &#181;L of nuclease-free water.</li>
	<li>Run the qPCR using primers targeting the promoters and enhancers known to be active in adipocytes according to the cycling conditions indicated in Table 7.</li>
	<li>Use negative control primers targeting closed/silent genomic regions (Table 7).</li>
	<li>Calculate the enrichment of the promoter/enhancers over the negative controls (Table 8). 
	&#8203;NOTE: It is expected to see &#8805;10-20-fold enrichments with successful samples.</li>
</ol>
14. Sequencing
<ol>
	<li>Submit the ATAC-seq libraries for sequencing. Use paired-end sequencing (2 x 34 bp, 2 x 50 bp or longer) with average read numbers of 10-30 million reads per sample.</li>
</ol>

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The authors declare that they have no relevant or material financial interests relating to the research described in this paper.

In this paper, we have presented an optimized ATAC-seq protocol to assess adipocyte-specific chromatin accessibility in vivo. This ATAC-seq protocol using the Adipoq-NuTRAP mouse successfully generated adipocyte-specific chromatin accessibility profiles. The most critical factor for successful and reproducible ATAC-seq experiments is nucleus quality. It is critical to immediately snap-freeze the dissected adipose tissues in liquid nitrogen and store them safely at &#8722;80 &#176;C without thawing until use. It ...

Adipose tissue, which is specialized for storing excess energy in the form of lipid molecules, is a key organ for metabolic regulation. The strict control of adipocyte formation and maintenance is vital for adipose tissue function and whole-body energy homeostasis1. Many transcriptional regulators play a critical role in the control of adipocyte differentiation, plasticity, and function; some of these regulators are implicated in metabolic disorders in humans2,3. Recent advances in high-throughput sequencing techniques for gene expression and epigenomic analysis have further facilitated the discovery of the molecular regulators of adipocyte biology4. Molecular profiling studies using adipose tissues are challenging to conduct due to the heterogeneity of these tissues. Adipose tissue consists primarily of adipocytes, which are responsible for fat storage, but also contains various other cell types, such as fibroblasts, endothelial cells, and immune cells5. In addition, the cellular composition of adipose tissue is dramatically altered in response to pathophysiological changes such as temperature and nutritional status6. To overcome these problems, we previously developed a transgenic reporter mouse, named Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP), which produces GFP-tagged ribosomes and mCherry-tagged biotinylated nuclei in a Cre recombinase-dependent manner7. The dual-labeling system enables one to perform cell type-specific transcriptomic and epigenomic analysis with tissues. Using NuTRAP mice crossed with adipocyte-specific adiponectin-Cre lines (Adipoq-NuTRAP), we previously characterized gene expression profiles and chromatin states from pure adipocyte populations in vivo and determined how they are altered during obesity7,8. Previously, NuTRAP mice crossed with brown and beige adipocyte-specific Ucp1-Cre lines (Ucp1-NuTRAP) allowed us to characterize the epigenomic remodeling of the rare thermogenic adipocyte population, beige adipocytes, in response to temperature changes9.
ATAC-seq is a widely used analytical method to assess genome-wide chromatin accessibility.The hyper-reactive Tn5 transposase used in ATAC-seq allows for the identification of open chromatin regions by tagging sequencing adapters in the chromatin-accessible region of nuclei10. ATAC-seq is a simple method, yet it provides robust results and is highly efficient even with low-input samples. It has, thus, become one of the most popular epigenomic profiling methods and has contributed to the understanding of the regulatory mechanisms of gene expression within diverse biological contexts. Since the original ATAC-seq protocol was created, various ATAC-seq-derived techniques have been further developed to modify and optimize the protocol for various types of samples. For example, Fast-ATAC is designed for analyzing blood cell samples11, Omni-ATAC is an optimized protocol for frozen tissue samples12, and MiniATAC-seq is effective for early-stage embryo analysis13. However, applying the ATAC-seq method to adipocytes, especially from tissue samples, is still challenging. In addition to the heterogeneity of adipose tissue, its high lipid content may interfere with efficient recombination reactions by Tn5 transposase even after nucleus isolation. Furthermore, the high mitochondrial content in adipocytes, particularly in brown and beige adipocytes, causes high mitochondrial DNA contamination and wasted sequencing reads. This paper describes a protocol for adipocyte-specific ATAC-seq using Adipoq-NuTRAP mice (Figure 1). By taking advantage of fluorescence-labeled nucleus sorting, this protocol allows the collection of pure populations of adipocyte nuclei away from other confounding cell types and the efficient removal of lipids, mitochondria, and tissue debris. Hence, this protocol can generate cell type-specific high-quality data and minimize waste from mitochondrial reads while using a reduced amount of input and reagents compared to the standard protocol.

<table><tbody><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adiponectin-Cre mouse</td><td>The Jackson Laboratory</td><td>28020</td><td></td></tr><tr><td>NuTRAP mouse</td><td>The Jackson Laboratory</td><td>29899</td><td></td></tr><tr><td>&lt;strong&gt;Reagents &amp;amp; Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5 mL DNA-LoBind tubes</td><td>Eppendorf</td><td>86-923</td><td></td></tr><tr><td>100 &amp;micro;m cell strainer</td><td>Falcon</td><td>352-360</td><td></td></tr><tr><td>15 mL tubes</td><td>VWR</td><td>525-1071</td><td></td></tr><tr><td>2x TD buffer</td><td>Illumina</td><td>15027866</td><td></td></tr><tr><td>384-well PCR plate</td><td>Applied biosystem</td><td>4483285</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Falcon</td><td>352-340</td><td></td></tr><tr><td>50 mL tubes</td><td>VWR</td><td>525-1077</td><td></td></tr><tr><td>AMPure XP reagent (SPRI beads)</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Bioanalyzer High Sensitivity DNA kit</td><td>Agilent Technologies</td><td>5067-4626</td><td></td></tr><tr><td>Clear adhesive film</td><td>Applied biosystem</td><td>4306311</td><td></td></tr><tr><td>DNase/RNase-free distilled water</td><td>Invitrogen</td><td>10977015</td><td></td></tr><tr><td>Dounce tissue grinder</td><td>DWK Life Sciences</td><td>357542</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779</td><td></td></tr><tr><td>DynaMag-96 side skirted magnet</td><td>Thermo Fishers</td><td>12027</td><td></td></tr><tr><td>FACS tubes</td><td>Falcon&amp;nbsp;</td><td>28719128</td><td></td></tr><tr><td>HEPES</td><td>Boston BioProducts</td><td>BBH-75</td><td></td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>2134015</td><td></td></tr><tr><td>KCl (2 M)</td><td>Boston BioProducts</td><td>MT-252</td><td></td></tr><tr><td>Magnetic separation rack for PCR 8-tube strips</td><td>EpiCypher</td><td>10-0008</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2 &lt;/sub&gt;(1 M)</td><td>Boston BioProducts</td><td>MT-200</td><td></td></tr><tr><td>MinElute PCR purification kit</td><td>Qiagen</td><td>28004</td><td></td></tr><tr><td>NEBNext High-Fidelity 2x PCR master mix</td><td>BioLabs</td><td>M0541S</td><td></td></tr><tr><td>NP40</td><td>Thermo Fishers</td><td>28324</td><td></td></tr><tr><td>PCR 8-tube strip</td><td>USA scientific</td><td>1402-4708</td><td></td></tr><tr><td>Protease inhibitor cocktail (100x)</td><td>Thermo Fishers</td><td>78439</td><td></td></tr><tr><td>Qubit dsDNA HS assay kit</td><td>Invitrogen</td><td>Q32851</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389-1KG</td><td></td></tr><tr><td>SYBR Green I (10,000x)</td><td>Invitrogen</td><td>S7563</td><td></td></tr><tr><td>TDE I enzyme</td><td>Illumina</td><td>15027865</td><td></td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td>FACSAria Fusion</td><td></td></tr><tr><td>Qubit fluorometer</td><td>Invitrogen</td><td>Q33226</td><td></td></tr><tr><td>Real-Time PCR system</td><td>Thermo Fishers</td><td>QuantStudio?5</td><td></td></tr></tbody></table>

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.

To analyze adipose tissue using this ATAC-seq protocol, we generated Adipoq-NuTRAP mice that were fed chow diets; we then isolated adipocyte nuclei from epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and brown adipose tissue (BAT) by using flow cytometry. The isolated nuclei were used for tagmentation, followed by DNA purification, PCR amplification, quality check steps, sequencing, and data analysis, as described above. The purpose of this representative experiment was to profile the chrom...

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

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 ...

adipocyte-specific-atac-seq-with-adipose-tissues-using-fluorescence

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

2.1K Views.  Indiana University School of Medicine. 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.

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

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Developmental Biology

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 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.

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 ...

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 ...

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

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Biology

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

Medicine

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 ...

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium (2H)-Labeling Approach

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat mass or distribution are regulated by changes in the synthesis and breakdown (i.e., turnover) of adipose cells and triacylglycerols. Evidence suggests that the manner and magnitude of subcutaneous adipose tissue expansion (i.e., hypertrophy vs. hyperplasia) and turnover can influence metabolic health, as adipogenesis has been implicated in the pathogenesis of obesity and related diseases. Despite the potential role of adipose turnover in human health, there is a lack of knowledge about the in vivo kinetics of adipose cells. This is due, in part, to the slow turnover rate of the cells in adipose tissue and the practical complexity of directly labeling their metabolic precursors in vivo. Herein, we describe methods to measure in vivo adipose kinetics and turnover rates in humans through the consumption of deuterium (2H)-labeled water. The incorporation of 2H into the deoxyribonucleotide moieties of DNA in pre-adipocytes and adipocytes provides an accurate measure of cell formation and death (adipose turnover). Overall, this is an innovative approach to measuring in vivo adipose kinetics and represents a substantive departure from other in vitro assessments.

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium 2H-Labeling Approach

Presented here is a protocol to measure in vivo adipose tissue kinetics in humans using the deuterium (2H)-labeling method.

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat ...

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

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Immunomagnetic Separation of Fat Depot-specific Sca1^high Adipose-derived Stem Cells (ASCs)