The animal handling steps of the protocol have been approved by Boston University&#8217;s Institutional Animal Care and Use Committee (IACUC).
1. Day 1: Dissection and Preparation of BAT for Chromatin Immunoprecipitation (ChIP)
<ol>
	<li>Euthanize mice using a carbon dioxide (CO2) chamber, and perform the dissection immediately afterwards. Spray mouse fur with 70% ethanol before incision. Place the mouse with its back facing up, then cut open the skin along the neck. Locate the BAT directly under the skin between the shoulders (interscapular; it appears as two lobes, butterfly-shaped, with a thin layer of white fat that needs to be carefully removed7). 
	NOTE: For a 3 month-old C57BL/6J mouse that is fed a regular chow diet (mouse weight at this age ranges between 27 and 30 g), each BAT lobe is approximately 1 cm long. The total weight of the BAT depot is approximately 0.15 g in both male and female mice.</li>
	<li>Use 1 BAT pad for 3 ChIPs. Multiple BAT pads can be simultaneously processed until sonication.</li>
	<li>Crosslink each BAT pad in 10 mL of phosphate-buffered saline (PBS) containing 1% formaldehyde for 15 min rotating at 150 rpm at room temperature (RT).</li>
	<li>Quench crosslinking by adding 2.5 M glycine to a final concentration of 125 mM for 5 min, rotating at 150 rpm at RT.</li>
	<li>Transfer the BAT pad to 500 &#181;L of hypotonic lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF) and add 2 stainless steel beads (size: 3.2 mm diameter, see Table of Materials) for each BAT pad. Use a bead-based tissue homogenizer (see Table of Materials) to shred the tissue.</li>
	<li>Start with 5 min at max power. If needed, extend the time until the tissue is homogenized and single cells are separated. Transfer the samples into a new tube and centrifuge at 4 &#176;C at 16,000 x g for 10 min.</li>
	<li>After centrifugation, transfer the supernatant into a new tube and keep the cell pellet on ice. Centrifuge the supernatant at 4 &#176;C at 16,000 x g for 10 min to prevent loss of floating cells. Discard the final supernatant and resuspend both cell pellets together in 300 &#181;L of SDS lysis buffer (SDS 1%; EDTA 10 mM; Tris-HCl pH 8, 50 mM) with 1x protease inhibitor (see Table of Materials). Incubate on ice for 10 min.</li>
	<li>Sonicate the lysates to generate DNA fragments 200&#8211;500 base pairs long. After sonication, remove debris by centrifugation for 10 min at 16,000 x g at 4 &#176;C and check the success of sonication via DNA electrophoresis on a 1% agarose gel. For a medium-size gel, run at 120 V; a good separation is achieved after 45 min. 
	NOTE: Sonication optimization may be required to achieve proper size of fragments. With the chosen sonicator (see Table of Materials), this requires 2 times at 320 W output power and 20 KHz frequency for 10 min, using cycles of 30 s on/30 s off.</li>
	<li>Dilute the supernatant fraction 10-fold (final volume is 3 mL for each BAT pad) in ChIP dilution buffer (SDS 0.01%; Triton 1.1%; EDTA 1.2 mM; Tris-HCl pH 8, 16.7 mM; NaCl 167 mM) with 1x protease inhibitor. Keep aside 1% of this chromatin solution (equal to 30 &#181;L for 3 mL) as the ChIP input. Keep the inputs overnight at 4 &#176;C (until step 2.4). 
	Note: The above fraction will provide the input reference for relative quantification of immunoprecipitated material compared to the amount of DNA present in each sample prior to immunoprecipitation.</li>
	<li>Add the ChIP antibody (see Table of Materials for the antibodies used here as an example) to 1 mL of chromatin solution and incubate overnight at 4 &#176;C with rotation. Perform parallel immunoprecipitation with corresponding pre-immune IgG, if available, or normal IgG of the same species (e.g., regular rabbit IgG if the antibody is produced in a rabbit). Alternatively, save 1 mL of chromatin solution for a no-antibody control. 
	NOTE: The optimal amount of antibody should be determined experimentally for each new antibody, if the antibody concentration is not known. Otherwise, 2&#8211;5 &#181;g of antibody is a reasonable starting amount to use. Use ChIP-grade antibodies whenever possible, or antibodies that have been validated for immunoprecipitation.</li>
</ol>
2. Day 2: Collection of the Immune Complexes
<ol>
	<li>Add 50 &#181;L of Protein A Agarose 50% slurry to the immune complexes and incubate for 1 h at 4 &#176;C with rotation at 150 rpm.</li>
	<li>Pellet the beads by centrifugation for 1 min at 100 x g at 4 &#176;C. Perform sequential washes of the beads with buffers of increasing stringency.
	<ol>
		<li>First, wash with low-salt wash buffer (150 mM NaCl; Tris-HCl pH 8, 20 mM; EDTA 2 mM; Triton-X 1%; SDS 0.1%), then with high-salt wash buffer (500 mM NaCl; Tris-HCl pH 8, 20 mM; EDTA 2 mM, Triton-X1%; SDS 0.1%), then with LiCl wash (0.25 M LiCl; Tris-HCl pH 8, 10 mM; EDTA 1 mM; Igepal 1%; deoxicholate 1%), and finally with 1x TE (Tris-HCl pH 8, 10 mM; EDTA).</li>
		<li>Perform all washes on 0.45 &#181;m PVDF centrifugal filter columns (Table of Materials) by first transferring the beads in the columns using 500 &#181;L of low-salt wash buffer. Pellet the beads in the columns for 3 min at 2,500 x g. Discard the flow-through and repeat 1 time for all the wash buffers listed in step 2.2.1.</li>
	</ol>
	</li>
	<li>After the washes are completed, elute the immune complexes by adding 300 &#181;L of elution buffer 1% SDS, 0.1 M NaHCO3) to the pelleted beads in the columns. Incubate at RT for 30 min on a shaker at 400 rpm. Collect the eluate by spinning down columns for 3 min at 2,500 x g in a fresh tube.</li>
	<li>Reverse the crosslinks by incubating the eluate and inputs collected in step 1.9 at 65 &#176;C overnight.</li>
</ol>
3. Day 3: Recovery of DNA with Phenol/Chloroform Extraction
<ol>
	<li>Add 300 &#181;L of phenol:chloroform:IAA, 1:1 to the eluate and inputs. Vortex for 10 s.</li>
	<li>Spin at 4 &#176;C for 15 min at 21,000 x g. Transfer the supernatant to a fresh tube. Add 3 &#181;L of glycogen, 30 &#181;L of 3 M sodium acetate, and 750 &#181;L of cold 100% ethanol. Vortex for 10 s. Store at -80 &#176;C for 2 h.</li>
	<li>Spin down at 21,000 x g at 4 &#176;C for 20 min, keep the pellet, and discard the supernatant. Wash pellet with 1 mL of cold 70% EtOH, then spin down at 21,000 x g at 4 &#176;C for 5 min. Discard the supernatant and air-dry the pellet.</li>
	<li>Resuspend the pellet in 70 &#181;L of DNAse-free water.</li>
	<li>Proceed to step 4 for testing chromatin occupancy on specific genomic regions by quantitative polymerase chain reaction (qPCR) or to step 5 to prepare samples for sequencing.</li>
</ol>
4. Analysis of ChIP using qPCR (Single/Multiple Genes Readout)
<ol>
	<li>Dilute the input DNA from step 3.4 to generate a standard reference curve. Serial dilutions of 1/10, 1/100, and 1/1000 are usually sufficient to capture the linear range, but further dilutions may be required for more concentrated samples.</li>
	<li>For each primer pair (here, we have used primers targeting NDUFV1 and TOMM20 promoters), prepare two sets of qPCR reactions: one using the diluted input samples from step 4.1, and a second with the DNA samples isolated by immunoprecipitation from step 3.4. Perform each reaction in triplicate. 
	NOTE: Reactions can be set up in parallel for multiple genes using 96- or 384-well plates.</li>
	<li>Set up a reaction volume of 10 &#956;L per well with 5 &#956;L of 2x PCR mix (Table of Materials), 1 &#956;L of primer mix (1 &#956;M forward primer and 1 &#956;M reverse primer), and 4 &#956;L of DNA from ChIP (or the input control).</li>
	<li>Briefly spin down the plate.</li>
	<li>Run the qPCR reactions on a real-time PCR system using the protocol recommended by the manufacturer for reagent detection (Table of Materials). Here we have used the following conditions: 1) 1s at 95&#176;C for 1 cycle; 2) 1 s at 95 &#176;C followed by 20 s at 60 &#176;C for 45 cycles; 3) 15 s at 95 &#176;C followed by 60 s at 60 &#176;C followed by 15 s at 95 &#176;C for 1 cycle. 
	NOTE: Check the dissociation curve: the presence of one peak in the thermal dissociation plot suggests a single amplicon is generated from the PCR reaction. If more than one peak is visualized, this is potentially indicative of multiple, non-specific amplicons; in which case, we recommend the design of alternative primer pairs.</li>
	<li>Determine the linear phase of exponential amplification of the PCR reaction for each primer set by calculating the standard curve using the series of diluted genomic DNA in step 4.1. according to the Ct (cycle threshold) value.</li>
	<li>If the Ct value of DNA from ChIP and input control are within the linear range of Ct value, calculate the enrichment of DNA from ChIP relative to the input, according to the dilution factor of DNA from ChIP and input control in step 4.1.</li>
</ol>
5. Amplification of DNA from ChIP for High-throughput Sequencing (Genome-wide Readout)
NOTE: This step can be outsourced to an academic core facility or commercial sequencing company when sequencing capabilities are not available in-house.
<ol>
	<li>Prepare a DNA library from the extracted DNA with an appropriate ChIP library preparation kit (see Table of Materials) following the manufacturer&#8217;s instructions.</li>
	<li>Sequence the library on a high-throughput sequencing system, using 50 bp single-end as readout (see Table of Materials). 
	NOTE: According to ChIP-seq guidelines suggested by ENCODE6, a minimum of 10 million reads is recommended for mammalian genomes.</li>
</ol>
6. Raw Data Analysis
<ol>
	<li>Perform quality control on raw sequencing reads using FastQC8. 
	NOTE: Common sequencing quality metrics include per base sequence quality, per base sequence content, per sequence GC content, sequence length, and sequence duplication levels. Generally, a read quality score of Q &#62; 30 across each base position is indicative of high-quality sequencing results. The distribution of GC content across all reads should roughly resemble a normal distribution, with a peak centered around the species&#8217; actual genomic GC content percentage.</li>
	<li>Remove any sequencing adapter contamination and low-quality reads through the read trimming utility Trimmomatic9. 
	NOTE: The implemented default parameters specify the removal of platform dependent adapters, removal of leading and trailing low-quality bases, and use of a 4-base wide sliding window to trim reads when the average quality per base drops below a specified threshold.</li>
	<li>Align processed reads to the mouse MM10 reference genome10 with the Bowtie2 aligner11 using default parameters.</li>
	<li>Filter aligned reads by removing those with a Bowtie2 mapping quality score (MAPQ) of less than 10 using Samtools12.</li>
	<li>Determine various post-alignment quality metrics such as percent reads mapped, strand cross-correlation, cumulative read coverage, and sample replicate reproducibility&#173;. 
	Note: Various packages including SPP13, ChIPqc14, and Deeptools15 are available to calculate these quality metrics.</li>
	<li>Perform peak calling analysis with an input control or appropriate KO sample using the MACS algorithm (MACS2)16 with default parameters.</li>
	<li>Remove any called peaks that overlap with known blacklisted regions17 from the mm10 annotation using Bedtools18. 
	NOTE: Blacklisted regions are areas in the genome that have been shown to have high signal or read counts independent of cell line or sequencing technique. These regions display artificially high artifact signals in certain regions of the genome that can be filtered out of functional genomic sequencing experiments. All replicates should be processed independently through the pipeline and may then be used for irreproducibility discovery rate (IDR). IDR is employed to measure the reproducibility of findings between replicates through the method as described by Li, Brown, Huang, and Bickel19,20. The IDR method quantitatively assesses consistency between replicates and is recommended by ENCODE and modENCODE as part of their ChIP-seq guidelines.</li>
	<li>Use statistically significant peaks for various downstream analyses such as gene ontology21,22, enrichment, or motif analysis23.</li>
</ol>

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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=Histone+deacetylase+3+prepares+brown+adipose+tissue+for+acute+thermogenic+challenge.">Histone deacetylase 3 prepares brown adipose tissue for acute thermogenic challenge.</a> Nature. , (2017).</li><li>Harms, M. 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=PRDM16+binds+MED1+and+controls+chromatin+architecture+to+determine+a+brown+fat+transcriptional+program.">PRDM16 binds MED1 and controls chromatin architecture to determine a brown fat transcriptional program.</a> Genes &amp; Development. , (2015).</li><li>Shapira, S. N., 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=EBF2+transcriptionally+regulates+brown+adipogenesis+via+the+histone+reader+DPF3+and+the+BAF+chromatin+remodeling+complex.">EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex.</a> Genes &amp; Development. , (2017).</li><li>Landt, S. G., 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=ChIP-seq+guidelines+and+practices+of+the+ENCODE+and+modENCODE+consortia.">ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.</a> Genome Research. 22, 1813-1831 (2012).</li><li>Liisberg Aune, U., Ruiz, L., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Differentiation+of+Stromal+Vascular+Cells+to+Beige/Brite+Cells.">Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells.</a> Journal of Visualized Experiments. , e50191 (2013).</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30, 2114-2120 (2014).</li><li>. Genome Browser Gateway Available from: <a target="_blank" href="https://genome.ucsc.edu/cgi-bin/hgGateway?db=mm10">https://genome.ucsc.edu/cgi-bin/hgGateway?db=mm10</a> (2018)</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. , 357-359 (2012).</li><li>Li, H., 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=The+Sequence+alignment/map+(SAM)+format+and+SAMtools.">The Sequence alignment/map (SAM) format and SAMtools.</a> Bioinformatics. 25 (16), 2078-2079 (2009).</li><li>Kharchenko, P. V., Tolstorukov, M. Y., Park, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+analysis+of+ChIP-seq+experiments+for+DNA-binding+proteins.">Design and analysis of ChIP-seq experiments for DNA-binding proteins.</a> Nature Biotechnology. 26, 1351-1359 (2008).</li><li>Carroll, T. 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The protocol described here represents a valuable tool for performing ChIP from murine tissues, specifically optimized for brown adipose tissue. One of the greater challenges in performing ChIP from tissue is recovering a sufficient number of cells during sample preparation. Shearing the BAT using a tissue homogenizer blender coupled with stainless steel beads instead of a canonical glass pestle significantly reduces the number of cells lost due to unbroken tissue. Moreover, homogenizing the tissue directly in a hypotoni...

While white adipose tissue (WAT) is specialized for energy storage, brown adipose tissue (BAT) dissipates energy in the form of heat due to its ability to convert carbohydrates and lipids into thermal energy via mitochondrial uncoupling1. Because of this specialized function, the BAT depot is required for maintenance of body temperature in physiological conditions and in response to cold exposure. While gene expression changes during BAT differentiation and upon thermogenic stress have been extensively studied in vivo and in vitro, the molecular mechanisms underlying these changes have been mostly dissected in immortalized cell lines and primary pre-adipocytes, with the exception of several in vivo studies2,3,4,5.
Regulation of specific gene expression programs through transcriptional regulation is achieved by coordinated changes in chromatin structure via various transcription factors and co-factors actions. Chromatin immunoprecipitation (ChIP) is a valuable molecular biology approach for investigating the recruitment of these factors to DNA and for profiling the associated changes in the chromatin landscape. Key factors for the success of ChIP experiments include optimizations of crosslinking conditions and chromatin shearing consistency throughout different samples, availability of adequate starting material, and, most notably, quality of the antibodies. When performing ChIP from whole tissues, it is also important to consider heterogeneity of the samples and optimize the protocol to improve efficiency of nuclei isolation, with the latter being a particularly sensitive step when working with adipose tissue due to the elevated lipid content. In fact, molecular isolation techniques from whole adipose depots are complicated by the presence of high levels of triglycerides, and protocols must be optimized to increase the amount of chromatin isolation. Finally, when high-throughput sequencing is performed after ChIP-DNA isolation, the sequencing depth is critical for determining the number of peaks that are confidently detected.
Here, we refer to the working standards and general guidelines for ChIP-seq experiments recommended by the ENCODE and modENCODE consortia6 for best practices, and we focus on a step-by-step description of a protocol optimized for ChIP-seq from BAT. The described protocol allows for efficient isolation of chromatin from adipose tissue to perform genome-wide sequencing for DNA-binding factors with well-defined peaks as well as histone marks with more diffuse signals.

<table><tbody><tr><td>Bullet Blender Tissue Homogenizer&amp;nbsp;</td><td>Next Advence&amp;nbsp;</td><td>BBX24</td><td></td></tr><tr><td>Stainless Steel Beads 3.2 mm Diameter</td><td>Next Advence&amp;nbsp;</td><td>SSB32</td><td></td></tr><tr><td>Bioruptor Sonicator</td><td>Diagenode</td><td></td><td></td></tr><tr><td>1.5 mL Micro Tube TPX Plastic&amp;nbsp;</td><td>Diagenode</td><td>C30010010-5</td><td></td></tr><tr><td>Complete-Protease inhibitor</td><td>Roche&amp;nbsp;</td><td>11836145001</td><td></td></tr><tr><td>Protein A Agarose Slurry&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>101041</td><td></td></tr><tr><td>GPS2 antibody</td><td>In house</td><td></td><td>Rabbit polyclonal, Ct antibody (Cardamone et al., Mol Cell 2018)</td></tr><tr><td>Pol2 antibody&amp;nbsp;</td><td>Diagenode</td><td>C15100055</td><td></td></tr><tr><td>h3K9me3 antibody</td><td>Millipore&amp;nbsp;</td><td>05-1242</td><td></td></tr><tr><td>Fast Syber Green Master Mix</td><td>Aplied Biosytem</td><td>4385612</td><td></td></tr><tr><td>ViiA7</td><td>Aplied Biosytem</td><td></td><td></td></tr><tr><td>TruSeq ChIP Library Preparation Kit</td><td>Illumina&amp;nbsp;</td><td>IP-202-1012</td><td></td></tr><tr><td>HiSeq 2000&amp;nbsp;</td><td>Illumina&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

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.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58682/58682fig1.jpg" /> 
Figure 1: ChIP validation by qPCR. ChIP-qPCR analysis of representative GPS2 target genes NDUFV1 (left) and TOMM20 (right) in the BAT of WT and GPS2-AKO mice, showing relative changes in the level of H3K9 methylation and GPS2 and Pol2 binding. The bar graphs represent the sample mean of 3 replicates with *p &#60; 0.05 and **p &#60; 0.01 as calculated with Student&#39...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation of Murine Brown Adipose Tissue at JoVE.com

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

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 ...

chromatin-immunoprecipitation-of-murine-brown-adipose-tissue

Research

JoVE Journal

Genetics

8.0K Views.  Boston University School of Medicine. De gemeten het wordt gebruikt voor het uitvoeren van efficiënte ChIP sequencing van zowel ester en niet-ester eiwitten in bruin lipide weefsel geïsoleerd van de muis. Het belangrijkste voordeel van de techniek is dat het protocol is geoptimaliseerd voor een efficiënte immunoprecipitatie van chromatine-geassocieerd complex uit het lipideweefsel. Voor incisie, spray de geëuthanaseerde muis met 70%ethanol.Plaats de muis met de rug naar boven gericht, maak dan een incisie langs de nek....

Video: Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Biochemistry

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

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

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

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

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

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Medicine

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol&#160;reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

This study describes a new method of isolating murine brown adipocytes for gene and protein expression analysis.

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs ...

Immunology and Infection

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose (FDG) after a meal or in obese or diabetic patients fails as the method of choice. The main reason is that 18F-FDG competes with the postprandial high glucose plasma concentration for the same glucose transporter on the membrane of BAT cells. In addition, BAT uses fatty acids as a source of energy as well, which is not visible with PET-CT and could be changed along with glucose concentration in obese and diabetic patients. Therefore, to estimate the physiological importance of BAT in animals and humans, a new infrared thermography method used in recent publications is applied.
After overnight fasting, BAT activity was measured by infrared thermography before and after a meal in human volunteers and&#160;female wild-type mice. The camera software calculates the object&#39;s temperature using distance from the object, skin emissivity, reflected room temperature, air temperature, and relative humidity. In mice, the shaved area above the BAT was a region of interest for which average and maximal temperatures were measured. The phase of the estrous cycle in female mice was determined after an experiment by vaginal smears stained with cresyl violet (0.1%) stain solution. In healthy volunteers, two skin areas of the neck were selected: the supraclavicular area (above the collarbone, where BAT cells are present) and the interclavicular area (between the collarbones, where there is no BAT tissue detected). BAT activity is determined by the subtraction of those two values. Also, the average and maximal temperatures of skin areas could be determined in animals and human participants.
Changes in BAT activity after a meal measured by infrared thermography, a non-invasive and more sensitive method, were shown to be sex, age, and phase of the estrous cycle dependent in laboratory animals. As part of diet-induced thermogenesis, BAT activation in humans was also proven to be sex, age, and body mass index dependent. Further determining the pathophysiological changes in BAT activity after a meal will be of great importance for participants with high glucose plasma concentrations (obesity and diabetes mellitus type 2), as well as in different laboratory animals (knock-out mice). This method is also a variable tool for determining possible activating drugs that could rejuvenate BAT activity.

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory animals.

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Biology

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on host and/or pathogen chromatin. Chromatin preparation from tissues creates additional challenges that need to be overcome to obtain reproducible and reliable protocols comparable to those used for cell culture. Tissue disruption and fixation are critical steps to achieve efficient shearing of chromatin. Coexistence of different cell types and clusters may also require different shearing times to reach optimal fragment size and hinders shearing reproducibility. The purpose of this method is to achieve reliable and reproducible host chromatin preparations from frozen tissue (liver) suitable for both ChIP-qPCR and next generation sequencing (NGS) applications. We observed that the combination of liquid nitrogen tissue pulverization followed by homogenization leads to increased reproducibility compared to homogenization only, since it provides a suspension consisting mostly of dissociated single cells that can be efficiently sheared. Moreover, the fixation step should be performed under mild rotation to provide homogeneous crosslinking. The fixed material is then suitable for buffer-based nuclei isolation, to reduce contamination of cytoplasmic protein and pathogen DNAs and RNAs (when applicable), avoiding time-consuming centrifugation gradients. Subsequent sonication will complete nuclear lysis and shear the chromatin, producing a specific size range according to the chosen shearing conditions. The size range should fall between 100 and 300 nt for NGS applications, while it could be higher (300-700 nt) for ChIP-qPCR analysis. Such protocol adaptations can greatly improve chromatin analyses from frozen tissue specimens.

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

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