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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animals</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>Reagents &#38; Materials</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 &#181;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 &#181;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&#160;</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>MgCl2 (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>Instruments</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&#8239;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...

Watch this Scientific Journal Video about Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting at JoVE.com

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

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

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL).

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

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 ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible chromatin. In this paper we present an improved ATAC-seq protocol that reduces contaminating mitochondrial DNA reads. While previous ATAC-seq protocols have struggled with an average of 50% contaminating mitochondrial DNA reads, the optimized&#160;lysis buffer introduced in this protocol reduces mitochondrial DNA contamination to an average of 3%. This improved ATAC-seq protocol allows for a near 50% reduction in the sequencing cost. We demonstrate how these high-quality ATAC-seq libraries can be prepared from activated CD4+ lymphocytes, providing step-by-step instructions from CD4+ lymphocyte isolation from whole blood through data analysis. This improved ATAC-seq protocol has been validated in a wide range of cell types and will be of immediate use to researchers studying chromatin accessibility.

Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.