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 BA...

<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. 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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. 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S., Liang, Z., Salama, R., Stark, R., de Santiago, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+artifact+removal+on+ChIP+quality+metrics+in+ChIP-seq+and+ChIP-exo+data.">Impact of artifact removal on ChIP quality metrics in ChIP-seq and ChIP-exo data.</a> Frontiers in Genetics. 5, 75 (2014).</li><li>Ram&#237;rez, F., 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=deepTools2:+A+next+Generation+Web+Server+for+Deep-Sequencing+Data+Analysis.">deepTools2: A next Generation Web Server for Deep-Sequencing Data Analysis.</a> Nucleic Acids Research. , (2016).</li><li>Zhang, Y., 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=Model-based+Analysis+of+ChIP-Seq+(MACS).">Model-based Analysis of ChIP-Seq (MACS).</a> Genome Biology. 9, R137 (2008).</li><li>ENCODE Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Quinlan, A. 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Irreproducible Discovery Rate (IDR) Available from: <a target="_blank" href="https://github.com/nboley/idr">https://github.com/nboley/idr</a> (2018)</li><li>The Gene Ontology Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+the+Gene+Ontology+Knowledgebase+and+Resources.">Expansion of the Gene Ontology Knowledgebase and Resources.</a> Nucleic Acids Research. 45, D331-D338 (2017).</li><li>Ashburner, M., 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=Gene+Ontology:+tool+for+the+unification+of+biology.">Gene Ontology: tool for the unification of biology.</a> Nature Genetics. 25, 25-29 (2000).</li><li>Boeva, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Genomic+Sequence+Motifs+for+Deciphering+Transcription+Factor+Binding+and+Transcriptional+Regulation+in+Eukaryotic+Cells.">Analysis of Genomic Sequence Motifs for Deciphering Transcription Factor Binding and Transcriptional Regulation in Eukaryotic Cells.</a> Frontiers in Genetics. 7, 24 (2016).</li></ol>

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.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Bullet Blender Tissue Homogenizer&#160;</td>
			<td style="width:113px;">Next Advence&#160;</td>
			<td style="width:119px;">BBX24</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Beads 3.2mm Diameter</td>
			<td style="width:113px;">Next Advence&#160;</td>
			<td>SSB32</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bioruptor Sonicator</td>
			<td>Diagenode</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 ml Micro Tube TPX Plastic&#160;</td>
			<td>Diagenode</td>
			<td style="width:119px;">C30010010-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Complete-Protease inhibitor</td>
			<td style="width:113px;">Roche&#160;</td>
			<td>11836145001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein A Agarose Slurry&#160;</td>
			<td style="width:113px;">Invitrogen&#160;</td>
			<td style="width:119px;">101041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">GPS2 antibody</td>
			<td style="width:113px;">In house</td>
			<td style="width:119px;"></td>
			<td>Rabbit polyclonal, Ct antibody (Cardamone et al., Mol Cell 2018)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Pol2 antibody&#160;</td>
			<td>Diagenode</td>
			<td style="width:119px;">C15100055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">h3K9me3 antibody</td>
			<td style="width:113px;">Millipore&#160;</td>
			<td style="width:119px;">05-1242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Fast Syber Green Master Mix</td>
			<td style="width:113px;">Aplied Biosytem</td>
			<td style="width:119px;">4385612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">ViiA7</td>
			<td style="width:113px;">Aplied Biosytem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq ChIP Library Preparation Kit</td>
			<td style="width:113px;">Illumina&#160;</td>
			<td>IP-202-1012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">HiSeq 2000&#160;</td>
			<td style="width:113px;">Illumina&#160;</td>
			<td style="width:119px;"></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 ...

The metered it's used for to perform efficient ChIP sequencing of both ester and non-ester proteins in brown lipid tissue isolated from mouse. The main advantage of the technique is that the protocol has been optimized for efficient immunoprecipitation of chromatin-associated complex from the lipid tissue. Before incision, spray the euthanized mouse with 70%ethanol.Place the mouse with its back facing up, then make an incision along the neck. Next, located BAT directly under the skin between the shoulders and carefully remove the thin layer of white fat. Cross-link each BAT pad in 10 milliliters of PBS containing 1%formaldehyde for 15 minutes at 150 RPM at room temperature.After 15 minutes, quench cross-linking by adding 2.5 molar glycine to the BAT, resulting in a final concentration of 125 millimolar. Place it on a shaker for five minutes, rotating at 150 RPM at room temperature. Subsequently, transfer the BAT pad to 500 microliters of hypotonic lysis buffer and add two stainless steel beads for each BAT pad.Then, use a bead-based tissue homogenizer to shred the tissue. Start with five minutes at max power. If needed, extend the time until the tissue is homogenized and single cells are separated.Transfer the sample into a new tube, and centrifuge at four degrees Celsius at 16, 000 G for 10 minutes. After 10 minutes, transfer the supernatant into a new tube and keep the cell pellet on ice. Centrifuge the supernatant at four degrees Celsius at 16, 000 G for 10 minutes to prevent loss of floating cells.Then, discard the final supernatant and re-suspend both cell pellets together in 300 microliters of SDS lysis buffer with one times protease inhibitor. Incubate on ice for 10 minutes. Next, sonicate the lysates to generate DNA fragments 200 to 500 base pairs long.After sonication, remove debris by centrifugation for 10 minutes at 16, 000 G at four degrees celsius. To perform immunoprecipitation, keep 1%of the chromatin solution aside as the ChIP input and keep the inputs overnight at four degrees Celsius. Next, add the ChIP antibody to one milliliter of chromatin solution and perform parallel immunoprecipitation with corresponding pre-immune IgG or normal IgG of the same species.Alternatively, save one milliliter of chromatin solution for a no antibody control. Incubate overnight at four degrees Celsius with rotation. To collect the immune complexes, add 50 microliters of protein A agarose 50%slurry to the immune complexes and incubate for one hour at four degrees Celsius with rotation at 150 RPM.After an hour, pellet the beads by centrifugation for one minute at 1000 times G at four degrees Celsius. Perform sequential washes of the beads on 0.45 micrometer PVDF centrifugal filter columns with buffers of increasing stringency by first transferring the beads in the columns with 500 microliters of low salt wash buffer. Then, pellet the beads in the columns for three minutes at 2500 G.Discard the flow-through.Repeat the wash with high salt wash buffer according to the low salt wash procedures. Then, repeat the procedures with lithium chloride and finally with one times TE.After the washes are completed, elute the immune complexes by adding 300 microliters of elution buffer to the pelleted beads in the columns. Incubate them at room temperature for 30 minutes on a shaker at 400 RPM.Subsequently, collect the elute by spinning down the columns for three minutes at 2500 G in a fresh tube. Reverse the cross-links by incubating the elute and inputs collected at 65 degrees Celsius overnight. To recover DNA, add 300 microliters of phenol chloroform IAA to the elute and inputs at a one-to-one ratio and vortex for 10 seconds.Next, spin down at 21, 000 G at four degrees Celsius for 20 minutes. Keep the pellet and discard the supernatant. Wash the pellet with one milliliter of cold 70%ethanol.Then spin down at 21, 000 G at four degrees Celsius for five minutes. Discard the supernatant and air dry the pellet. Re-suspend the pellet in 70 microliters of DNAse-free water for further procedures.Here is an example of ChIP Q PCR using BAT isolated from wildtype or GPS2-A knockout mice. Since GPS2 has been found to be required for the expression of nuclear encoded mitochondrial genes in different cell lines, the recruitment of GPS2 was tested on two specific nuclear encoded mitochondrial genes. NDUFV1 and TOMM20 in BAT from mice as the examples of a tissue highly enriched in mitochondria.GPS2 promoter occupancy was compared in GPS2-A knockout mice and wild type letter mates. An expected decrease was observed in GPS2 binding on selective target genes in the BAT from GPS2-A knockout mice. Using the protocol described here, an increased binding of RNA polymerase two and the repressive histone mark, H3K9ME3, was recorded in the BAT from GPS2-A knockout mice as compared to wild type letter mates.These results confirm the recruitment of GPS2 on selected nuclear encoded mitochondrial genes and show that GPS2 is required for preventing the accumulation of H3K9ME3 and thus required for stalling of pole two transcriptional activity on target promoters. The protocol described here, even if specifically optimized for the brown adipose tissue, represents a valued tool for performing ChIP from other murine and human tissue. Don't forget that working with phenochloroform can be extremely hazardous.Therefore, it's extremely important to always operate under the fume hood while using this reagent.

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Genetics

8.0K visualizações.  Boston University School of Medicine. O medido é usado para realizar sequenciamento chip eficiente de proteínas éster e não-éster em tecido lipídico marrom isolado do rato. A principal vantagem da técnica é que o protocolo foi otimizado para uma imunoprecipitação eficiente do complexo associado à cromatina a partir do tecido lipíduo. Antes da incisão, pulverize o rato eutanizado com 70% de etanol.Coloque o mouse de costas para cima e faça uma incisão ao longo do pescoço. Em seguida, localizado BAT diretamen...