Name: adipocyte-specific atac-seq with adipose tissues using fluorescence-activated nucleus sorting
Uploaded: 2023-03-17T14:00:00
Description: Watch this Scientific Journal Video about Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting at JoVE.com

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 ...

<ol>
	<li>Sethi, J. K., Vidal-Puig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+Adipocyte+biology.+Adipose+tissue+function+and+plasticity+orchestrate+nutritional+adaptation.">Thematic review series: Adipocyte biology. Adipose tissue function and plasticity orchestrate nutritional adaptation.</a> Journal of Lipid Research. 48 (6), 1253-1262 (2007).</li><li>Farmer, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+adipocyte+formation.">Transcriptional control of adipocyte formation.</a> Cell Metabolism. 4 (4), 263-273 (2006).</li><li>Bielczyk-Maczynska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White+adipocyte+plasticity+in+physiology+and+disease.">White adipocyte plasticity in physiology and disease.</a> Cells. 8 (12), 1507 (2019).</li><li>Basu, U., Romao, J. M., Guan, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipogenic+transcriptome+profiling+using+high+throughput+technologies.">Adipogenic transcriptome profiling using high throughput technologies.</a> Journal of Genomics. 1, 22-28 (2013).</li><li>Esteve Rafols, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue:+Cell+heterogeneity+and+functional+diversity.">Adipose tissue: Cell heterogeneity and functional diversity.</a> Endocrinologia y Nutricion. 61 (2), 100-112 (2014).</li><li>Kwok, K. H., Lam, K. S., Xu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+white+adipose+tissue:+Molecular+basis+and+clinical+implications.">Heterogeneity of white adipose tissue: Molecular basis and clinical implications.</a> Experimental and Molecular Medicine. 48, e215 (2016).</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>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=Adipocytes+fail+to+maintain+cellular+identity+during+obesity+due+to+reduced+PPAR&#947;+activity+and+elevated+TGF&#946;-SMAD+signaling.">Adipocytes fail to maintain cellular identity during obesity due to reduced PPAR&#947; activity and elevated TGF&#946;-SMAD signaling.</a> Molecular Metabolism. 42, 101086 (2020).</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=Warming+induces+significant+reprogramming+of+beige,+but+not+brown,+adipocyte+cellular+identity.">Warming induces significant reprogramming of beige, but not brown, adipocyte cellular identity.</a> Cellular Metabolism. 27 (5), 1121.e5-1137.e5 (2018).</li><li>Buenrostro, J. 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=Transposition+of+native+chromatin+for+fast+and+sensitive+epigenomic+profiling+of+open+chromatin,+DNA-binding+proteins+and+nucleosome+position.">Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.</a> Nature Methods. 10 (12), 1213-1218 (2013).</li><li>Corces, M. R., 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=Lineage-specific+and+single-cell+chromatin+accessibility+charts+human+hematopoiesis+and+leukemia+evolution.">Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution.</a> Nature Genetics. 48 (10), 1193-1203 (2016).</li><li>Corces, M. R., 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=An+improved+ATAC-seq+protocol+reduces+background+and+enables+interrogation+of+frozen+tissues.">An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.</a> Nature Methods. 14 (10), 959-962 (2017).</li><li>Wu, J., 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=Chromatin+analysis+in+human+early+development+reveals+epigenetic+transition+during+ZGA.">Chromatin analysis in human early development reveals epigenetic transition during ZGA.</a> Nature. 557 (7704), 256-260 (2018).</li><li>Bagchi, D. P., MacDougald, O. A. <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+dissection+of+diverse+mouse+adipose+depots.">Identification and dissection of diverse mouse adipose depots.</a> Journal of Visualized Experiments. (149), e59499 (2019).</li><li>So, J., 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=Chronic+cAMP+activation+induces+adipocyte+browning+through+discordant+biphasic+remodeling+of+transcriptome+and+chromatin+accessibility.">Chronic cAMP activation induces adipocyte browning through discordant biphasic remodeling of transcriptome and chromatin accessibility.</a> Molecular Metabolism. 66, 101619 (2022).</li><li>Loft, A., Herzig, S., Schmidt, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+GFP-tagged+nuclei+from+frozen+livers+of+INTACT+mice+for+RNA-+and+ATAC-sequencing.">Purification of GFP-tagged nuclei from frozen livers of INTACT mice for RNA- and ATAC-sequencing.</a> STAR Protocols. 2 (3), 100805 (2021).</li><li>Heyward, F. 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=Integrated+genomic+analysis+of+AgRP+neurons+reveals+that+IRF3+regulates+leptin's+hunger-suppressing+effects.">Integrated genomic analysis of AgRP neurons reveals that IRF3 regulates leptin's hunger-suppressing effects.</a> bioRxiv. , (2022).</li></ol>

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 ...

ATAC-seq is a powerful technique to understand gene expression regulatory mechanisms. However, adipose tissue is difficult using this technique because of its large lipid contents, high mitochondrial contamination, and cellular heterogeneity. In this protocol, adipocyte-specific ATAC-sequencing was performed using fluorescence-activated nucleus sorting, which generates high-quality data with minimal mitochondrial DNA contamination.This method can also be used in other tissues where cell-type-specific Cre mouse lines are available with nuclear labeling transgenic reporter lines. Begin the nucleus isolation by chilling the glass Dounce homogenizers on ice with one glass Dounce per sample. Then, add 7 milliliters of the NPB mix to each glass Dounce.Then, place 50 to 100 milligrams of frozen adipose tissue in the glass Dounce, and cut it into a few smaller pieces using a long pair of scissors. Stroke 10 times with a loose pestle for all the samples, and then 10 times with a tight pestle. Filter the content through a 100 micrometre strainer into a new 50 milliliter tube before transferring the filtered homogenates to a 15 milliliter tube for centrifugation.After removing the supernatant, resuspend the pellet in 500 microliters of PBS-N by gentle but thorough finger-tapping. Next, filter the suspension through a 40 micrometer strainer into a 50 milliliter tube and rinse the strainer with 250 microliters of PBS-N. Transfer the filtered nuclear suspension to a fluorescence-activated cell sorting or FACS tube using a micropipette.Prepare the collection tube by adding 500 microliters of PBS-N to the new 1.5 milliliter tube, and invert it a few times to wet all inner surfaces with the buffer. Briefly spin the tubes to bring down all the liquid before placing them on ice until sorting. For FACS, use the FSC-A/FSC-H gating for singlets and FSC-A/SSC-A for small or large debris removal.Gate for the mCherry/GFP-positive adipocyte nucleus population, and collect 10, 000 nuclei in each collection tube prepared earlier. For post-sorting nucleus preparation, add 500 microliters of PBS-N to each collection tube and mix it by inverting the tube a few times before centrifuging the collection tubes at 200 G for 10 minutes at 4 degrees Celsius. Completely remove the supernatant.To prepare 25 microliters of Tn5 master mixture, mix 12.5 microliters of 2x tagmentation DNA buffer or TD buffer, 1.25 microliters of Tn5 transposase or TDE I enzyme, and 11.25 microliters of nuclease-free water. Next, add 25 microliters of Tn5 master mixture to each nucleus pellet and resuspend it by gentle pipetting. Incubate at 600 RPM for 30 minutes at 37 degrees Celsius using a thermal mixer.Add 25 microliters of nuclease-free water to the 25 microliters of the mixture of Tn5 nucleus resuspension making the final volume of 50 microliters. Then, add 250 microliters of buffer PB and 5 microliters of 3 M sodium acetate with pH 5.2. After transferring the mixture to a column provided with the referenced kit, centrifuge it at 17, 900 G for 1 minute at room temperature.After discarding the flowthrough, place a spin column back in the same collection tube. Add 750 microliters of buffer PE containing absolute ethanol to the tube. Centrifuge the column, discard the flowthrough, and place the spin column back into the same collection tube.Centrifuge the tube, and discard the collection tube with the flowthrough. To amplify the transposed DNA fragments, prepare the PCR master mixture without adding a unique Ad2. n barcoding primer.Run the PCR. Run the quantitative PCR or qPCR. Check the amplification curves and identify the number of additional PCR cycles needed by estimating the number of cycles that reached approximately 35%of the maximum.Use the calculations to run the PCR for the remaining 45 microliters of the PCR reaction. Using the earlier procedures, elute the amplified DNA fragments with 20 microliters of buffer EB.Transfer the eluted DNA to a new PCR tube and adjust the volume to 100 microliters by adding 80 microliters of buffer EB.Next, add 55 microliters of SPRI beads and mix by pipetting. After 5 minutes of incubation at room temperature, separate it on a mini magnetic stand for 5 minutes.Once done, transfer 150 microliters of the supernatant to a new PCR tube, add 95 microliters of SPRI beads, and mix by pipetting. Incubate the reaction for 5 minutes before separating the beads on the mini magnetic stand. Discard the supernatant carefully and wash the beads for 1 minute using 200 microliters of 70%ethanol.Repeat two more washes. After the final wash, centrifuge the tubes at 1000 G for 1 minute. Pipette out the remaining ethanol and dry the pellet at 37 degrees Celsius for 2 minutes on a PCR machine with the lid open.Once dried, resuspend the pellet in 20 microliters of buffer EB by pipetting and incubate the tube for 5 minutes at room temperature. Separate the beads for 5 minutes on the mini magnetic stand before transferring 18 microliters of the supernatant containing the final library to a new 1.5 milliliter tube. mCherry-labeled and GFP-labeled adipocyte nuclei were distinguishable from the other cell types nuclei isolated from the adipose tissues of an Adipoq-NuTRAP mouse.Depending on the type of adipose depot, the difference is of approximately 50%in eWAT, 30%in iWAT, and 65%in BAT were observed in the adipocyte fractions. The samples requiring more than 15 PCR cycles indicated low-quality samples. The size distribution analysis of the ATAC-seq libraries demonstrated multiple peaks corresponding to the nucleosome-free region, or NFR, and mono, di, and multi-nucleosomes with average sizes of approximately 500 to 800 base pairs.Poor quality samples typically showed mostly NFR with no or few nucleosomal peaks. The quality check tests by the qRT-PCR analysis showed 10 to 20 fold enrichments with the positive genomic elements near adipocyte marker genes such as Adipoq, Fabp4, Plin1, and Pnpla2, while no enrichment was shown with the negative control. The enrichment with the thermogenic gene Ucp1 was observed specifically in BAT.The mitochondrial reads were less than 2%and the fraction under the peaks was approximately 18%to 44%Multiple strong peaks with high signal-to-noise ratios were revealed near adipocyte marker genes such as Adipoq, Fabp4, and Plin1 from all the adipose depots. Strong ATAC-seq peaks were observed at the brown adipocyte marker Ucp1 locus in the BAT sample. The most critical factor for this protocol is nucleus quality.It is important to use high-quality tissue samples and to gently handle the nuclei throughout the experiment, especially during resuspension after centrifugation and sorting. It's important to remember this protocol relies on transgenic reporter mice with nuclear labeling. NuTRAP mice were used here, but any similar system can be used.With our FACS data, one can perform computational multiple analysis to find key transcriptomic factors that regulate gene expression in biological settings.

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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 ...

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

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 ...

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 ...

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

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 ...

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 ...

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 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 ...