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>
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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. 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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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Genetics

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.