Name: Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells (Video) | JoVE Journal
Uploaded: 2010-07-29T17:00:00.000+00:00
Duration: 13 min 20 s
Description: Learn chromatin immunoprecipitation to study dynamic histone modifications during STAT1-activated gene expression in human cells. This method enables monitoring of transcription factor occupancy and chromatin changes.

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5 minutes

This work is supported by the University of Virginia, the University of Virginia Cancer Center and The Thomas F. and Kate Miller Jeffress Memorial Trust. We thank the James E. Darnell Lab (Rockefeller University) the Robert Ross Lab (Fordham University) for cell lines.

PLANNING
The day before a ChIP experiment is planned, cells should be cultured so that a sufficient number is available. Assume that each ChIP assay will require ~1-1.5 x 107 cells. Always plan to do both the negative (IgG) and positive (pan histone antibody) controls and duplicate experiments. 
 Tip - For 2FTGH cells, each ChIP assay requires about one 15 cm tissue culture dish at 80% confluency.
PART 1: PREPARE CHROMATIN AND PERFORM ChIP
<ol>
 <li>Remove culture media and induce cells for the times required with 20 mL of 10% cosmic calf serum (CCS)/DMEM containing IFN-gamma at 5 ng/mL. Replace the media on the uninduced plates with 20 mL of 10% CCS/DMEM as well.</li>
 <li>In a fume hood, add 540 μL 37% formaldehyde to 20 mL of media (1% final concentration) in the 15 cm dish. 
 Tip - Tilt plate and add the formaldehyde to the media pool - then rock plate to spread it evenly.
 </li>
 <li>Allow crosslinking to proceed for 10 minutes at room temperature.</li>
 <li>Add glycine to 0.125 M final concentration to stop crosslinking.</li>
 <li>Vacuum aspirate the media and wash cells once with 10 mL ice cold phosphate buffered saline (PBS).</li>
 <li>Collect cells with a cell scraper in ~ 7 mL PBS and combine cells (from 6 plates) that were treated identically in 50 mL conical tube on ice.</li>
 <li>Centrifuge (Beckman Coutler Allegra 12-R Centrifuge) at 1800 rpm at 4°C for 5 minutes and aspirate the PBS. 
 Tip - Snap freeze the cell pellet if stopping here and store at -80°C.</li>
 <li>Resuspend the cell pellet in 4 mL Swelling Buffer (25 mM HEPES pH 7.8, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP40. Add 1 mM DTT, Complete protease inhibitors and chill on ice prior to use) and transfer to a 15 mL conical tube. 
 Tip - This swelling buffer volume is appropriate for a 6 plate pooling (~6-9 x 107 cells). More or less buffer might change the douncing efficiency.
 </li>
 <li>Incubate the cell suspension on ice for 10 minutes.</li>
 <li>Transfer suspension to a pre-chilled 15 mL douncer (Wheaton) and dounce 20 strokes with pestle A.</li>
 <li>Transfer dounced cells back to a 15 mL conical tube on ice.</li>
 <li>Centrifuge at 2500 rpm for 5 minutes at 4°C and discard the supernatant.</li>
 <li>Resuspend the nuclear pellet (~ 200 μL from 6 plates) in 3 mL (15 pellet volumes) of Sonication Buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.1% SDS. Add Complete protease inhibitors and chill on ice just prior to use). 
 Tip - This sonication buffer volume is appropriate for a 6 plate pooling (~6-9 x 107 cells). More or less buffer might change the sonication efficiency.</li>
 <li>Sonicate nuclei suspension on ice for 10 cycles (20 seconds on/40 seconds off /amplitude setting of 8) using a Misonix Sonicator 3000 equipped with the microtip. This typically results in chromatin fragments averaging 200-1000 bps in size. 
 Tip - Because sonicators will vary, the sonication efficiency must be determined empirically. To do so, take aliquots of 20 μL before sonication and after each sonication cycle. Heat these samples at 65°C for 4 hours. Add DNA loading buffer and run a standard 1% agarose/TBE gel to visualize DNA fragments.) 
 </li>
 <li>Transfer sonicated material to an SS34 centrifuge tube and centrifuge for 20 minutes at 4°C at 13000 rpm in an SS34 rotor (Sorvall RC6 Plus Centrifuge) to pellet what is usually a small amount of cell debris.</li>
 <li>Carefully transfer the supernatant to a 15 mL conical tube on ice.</li>
 <li>Pre-clear by adding 360 μL Salmon Sperm DNA/Protein A or G agarose beads slurry and rocking at 4°C for at least 30 minutes. 
 Tip - This step decreases the noise in the assay that arises from non-specific binding events.</li>
 <li>Centrifuge at 1000 rpm at 4°C for 5 minutes to pellet the agarose beads.</li>
 <li>Transfer pre-cleared supernatant to another 15 mL conical tube on ice.</li>
 <li>Measure the A260 units of the pre-cleared chromatin and adjust the volume with Sonication Buffer to four (4) A260 units (0.2 mg/mL assuming 1 A260 unit = .050 mg/ml) 
 Tip - To measure the A260 units, dilute 10 μL of pre-cleared chromatin in 190 μL DDW; blank spectrophotometer with 10 μL sonication buffer in 190 μL DDW. 
 </li>
 <li>Save 75 μL of the chromatin (at 4 A260 units) as the Input Sample and store at 20°C until ready to reverse crosslinks. Tip - This step is crucial for data analysis. Do not neglect to take the input aliquot. </li>
 <li>Aliquot the prepared chromatin to 1.7 mL microfuge tubes on ice as 1 mL aliquots. Tip - If stopping here, snap freeze the prepared chromatin on dry ice and store at -80°C. NB: stored chromatin can degrade, producing less than optimal results, so avoid if possible. </li>
 <li>Add the ChIP-grade antibodies to the induced and uninduced samples sets in duplicate. Add IgG (2 μg) and pan Histone H3 antibody (15 μL) as the negative and positive controls to induced and uninduced samples sets. 
 Tip - The amount of antibody to use must be determined empirically or see the supplier's product sheet for suggested amounts. Typically 1-2 μg of a ChIP-grade antibody works well. The appropriate volume must be determined empirically for antibodies that are supplied as serum. 
 </li>
 <li>Rock all tubes overnight at 4°C.</li>
</ol>
PART 2: COLLECT IMMUNOCOMPLEXES
<ol>
 <li>Add 60 μL of Salmon Sperm DNA/Protein A or Protein G agarose beads slurry to all tubes. 
 Tip Use Protein A for rabbit polyclonal antibodies and Protein G for mouse monoclonal antibodies.</li>
 <li>Rock at 4°C for at least 60 minutes.</li>
 <li>Pellet the immunocomplexes (agarose beads) by microfuging gently at 2000 rpm at 4°C for 3 minutes.</li>
 <li>Aspirate the supernatant.</li>
 <li>Wash the immunocomplexes with 1 mL of each of the following Wash Buffers for 10 minutes with rocking at 4°C. Microfuge for 3 minutes at 2000 rpm at 4°C between each wash and aspirate supernatant. Keep samples chilled on ice. Add PMSF just prior to use.
 <ul>
 <li>Low Salt Wash - 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM PMSF</li>
 <li>High Salt Wash - 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.0, 500 mM NaCl, 1 mM PMSF</li>
 <li>LiCl Wash - 0.25 M LiCl, 1% NP-40, 1% Sodium deoxycholate, 1 mM EDTA, 10 mM Tris-Cl, pH 8.0, 1 mM PMSF</li>
 <li>TE Wash - TWO Washes with TE Buffer -10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 1 mM PMSF</li>
 </ul>
 </li>
 <li>Remove the final TE Buffer wash and to the beads, add 500 μL Elution Buffer (50 mM Tris-Cl, pH 8.0, 10 mM EDTA, 1% SDS). 
 Tip - The Elution Buffer should be prepared freshly. 
 </li>
 <li>Vortex to mix agarose beads in Elution Buffer and heat for 30 minutes at 65°C. 
 Tip - vortex each tube at least every 10 minutes during this 30 minutes.</li>
 <li>Microfuge for 30 seconds at 2000 rpm and transfer the eluent to a new microfuge tube. 
 Tip - During the elution, thaw your input samples and add 500 μL Elution Buffer to each one. Include these samples in all steps going forward. 
 </li>
 <li>Add 5 M NaCl to a final concentration of 200 mM to all samples and vortex.</li>
 <li>Incubate at 65°C overnight (or at least 4 hours).</li>
</ol>
PART 3: PURIFY ChIP'd AND INPUT DNA
<ol>
 <li>Microfuge all samples for 30 seconds to collect condensation on lids.</li>
 <li>Add 1μL of RNAse A (10 U/μL) to each tube and incubate at room temperature for 15 minutes.</li>
 <li>Add 10 μL 0.5 M EDTA, 40 μL 0.5 M Tris-Cl pH 6.5, 2 μL Proteinase K (10 mg/mL) and incubate at 45°C for 60 minutes. Tip Prepare a master mix of these reagents and add 52 μL to each tube.</li>
 <li>Phenol/chloroform/isoamyl alcohol extract and then chloroform/isoamyl alcohol extract the samples. </li>
 <li>Add 2 μL glycogen (25 μg/μL) and 0.1 volumes of 3M Na Acetate pH 5.2 and vortex.</li>
 <li>Add 2.5 volumes of 100% ethanol and vortex. </li>
 <li>Incubate at 20°C overnight.</li>
</ol>
PART 4: PURIFY ChIP'd AND INPUT DNA CONTINUED
<ol>
 <li>Microfuge samples for a minimum of 20 minutes at 13000 rpm at 4°C to precipitate the DNA/glycogen pellets.</li>
 <li>Wash with DNA/glycogen pellets with 1 mL of 70% Ethanol.</li>
 <li>Air dry the pellets or quickly dry in DNA Savant speed vacuum centrifuge. 
 Tip - Do not over dry the DNA.</li>
 <li>Resuspend DNA/glycogen pellets in 200 μL TE Buffer.</li>
 <li>DNA (1 μL) is ready for quantification via real-time PCR. Store remaining DNA at 20°C. 
 Tip - Using more than 1 μL of DNA can inhibit the real-time PCR reactions, so scale up with caution.</li>
</ol>
Part 5: REPRESENTATIVE RESULTS:
ChIP and Input DNA samples are quantified with Real-Time PCR 7,8 (Applied Biosystems 7500 Fast Real-Time PCR System) using primers specific to the gene locus of interest. Primers that target genomic sequence known to be enriched in or lacking in the histone modifications being assayed can be used as positive and negative controls as well. Data is presented as Percent of Input. The ChIP procedure should be wholly repeated with three biological replicates to ensure reported changes in histone modifications are statistically significant.
There are several important issues to consider when using real-time PCR to quantify DNA, including primer design, PCR efficiency, and the appropriate way to calculate the percent of input and normalization procedures. When profiling histone modification occupancy across a gene, the PCR efficiency must be consistent among all primer pairs. The real-time PCR system manufacturer can provide the necessary information and training.
Figure 1 shows representative results of chromatin immunoprecipitated during STAT1 activation of the IRF1 gene9, triggered by IFN-γ.
<img src="/files/ftp_upload/2053/2053fig1.jpg" alt="Gene expression analysis, H3K36me3, STAT1, Pol II, Pan H3; % input vs. primer location graph." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
 Figure 1. Occupancy of STAT1, RNA Polymerase II and H3K36me3 is dynamic during STAT1 activation of the IRF1 gene. (A, B, C) ChIP with α-H3K36me3, α-STAT1 and α-Pol II of chromatin collected from 2FTGH cells induced with IFN-γ for 30 minutes (red squares), 1.5 hours (blue circles), 5 hours (green triangles) or uninduced (black diamonds). IgG is the negative control (grey crosses). (D) Pan H3 is the positive control and shows the histone occupancy across the locus in the induced (blue circles) and uninduced conditions (black diamonds). The dip around -5 bp is due to the nucleosome depletion that is found at transcription start sites.

<ol><li>Levy, D., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signalling%3A+Stats%3A+transcriptional+control+and+biological+impact%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Signalling: Stats: transcriptional control and biological impact.</a> Nature Reviews Molecular Cell Biology. 3, 651-662 (2002).</li>
<li>Bromberg, J., Darnell, J. E. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+STATs+in+transcriptional+control+and+their+impact+on+cellular+function%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">The role of STATs in transcriptional control and their impact on cellular function.</a> Oncogene. 19, 2468-2473 (2000).</li>
<li>Hu, X., Ivashkiv, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cross-regulation+of+signaling+pathways+by+interferon-gamma%3A+implications+for+immune+responses+and+autoimmune+diseases%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases.</a> Immunity. 31, 539-550 (2009).</li>
<li>Campos, E. I., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histones%3A+annotating+chromatin%5BTitle%5D)+AND+%22Annu+Rev+Genet%22%5BJournal%5D)">Histones: annotating chromatin.</a> Annu Rev Genet. 43, 559-599 (2009).</li>
<li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+Modifications+and+Their+Function%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Chromatin Modifications and Their Function.</a> Cell. 128, 693-705 (2007).</li>
<li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translating+the+histone+code%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li>
<li>Wittwer, C. T., Herrmann, M. G., Moss, A. A., Rasmussen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+fluorescence+monitoring+of+rapid+cycle+DNA+amplification%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Continuous fluorescence monitoring of rapid cycle DNA amplification.</a> Biotechniques. 22 (130-131), 134-138 (1997).</li>
<li>Higuchi, R., Dollinger, G., Walsh, P. S., Griffith, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+amplification+and+detection+of+specific+DNA+sequences%5BTitle%5D)+AND+%22Biotechnology+%28N+Y%29%22%5BJournal%5D)">Simultaneous amplification and detection of specific DNA sequences.</a> Biotechnology (N Y). 10, 413-417 (1992).</li>
<li>Kroger, A., Koster, M., Schroeder, K., Hauser, H., Mueller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activities+of+IRF-1%5BTitle%5D)+AND+%22J+Interferon+Cytokine+Res%22%5BJournal%5D)">Activities of IRF-1.</a> J Interferon Cytokine Res. 22, 5-14 (2002).</li>
<li>Ross, R. A., Spengler, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+neuroblastoma+stem+cells%5BTitle%5D)+AND+%22Seminars+in+cancer+biology%22%5BJournal%5D)">Human neuroblastoma stem cells.</a> Seminars in cancer biology. 17, 241-247 (2007).</li>
</ol>

No conflicts of interest declared.

The ChIP protocol outlined has been used successfully in the lab to assay more than twenty different histone modifications. In addition, we have characterized changes in the occupancy of transcription factors, histone-modifying enzymes, proteins that recognize histone modifications and the RNA Polymerase II transcription machinery, at several IFN-&gamma; induced genes. We have also used the procedure to characterize changes in histone modifications during the differentiation of a cancer stem cell10. <p cla...

<table><tbody><tr><td>37% Formaldehyde</td><td>Fisher Scientific</td><td>BP531-500</td><td></td></tr><tr><td>Chloroform/Isoamyl alcohol</td><td>Acros Organics</td><td>AC32715-5000</td><td></td></tr><tr><td>Complete Mini Protease Inhibitors</td><td>Roche Group</td><td>1836153</td><td></td></tr><tr><td>Cosmic Calf Serum</td><td>Hyclone</td><td>SH30087.04N</td><td></td></tr><tr><td>DMEM</td><td>Hyclone</td><td>SH30243</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP118-500</td><td></td></tr><tr><td>Ethanol</td><td>Pharmco Products, Inc.</td><td>zp1110EP</td><td></td></tr><tr><td>Glycine</td><td>Roche Group</td><td>100149</td><td></td></tr><tr><td>Glycogen</td><td>Roche Group</td><td>901393</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3784</td><td></td></tr><tr><td>IFN-gamma</td><td>R&amp;amp;D Systems</td><td>285-IF-100</td><td></td></tr><tr><td>IgG</td><td>Jackson ImmunoResearch</td><td>109-005-003</td><td></td></tr><tr><td>KCl</td><td>MP Biomedicals</td><td></td><td></td></tr><tr><td>LiCl</td><td>Sigma-Aldrich</td><td>L4408</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Sodium Acetate</td><td>Fisher Scientific</td><td>BP333-500</td><td></td></tr><tr><td>Deoxycholic Acid Sodium Salt</td><td>Fisher Scientific</td><td>BP349-100</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>7647-14-5</td><td></td></tr><tr><td>NP40</td><td>US Biological</td><td>N3500</td><td></td></tr><tr><td>Anti-Histone H3, CT, pan</td><td>EMD Millipore</td><td>07-690</td><td></td></tr><tr><td>Phenol/chloroform/isoamyl</td><td>Fisher Scientific</td><td>108-95-2</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Fisher Scientific</td><td>7647-14-5</td><td></td></tr><tr><td>PMSF</td><td>EMD Millipore</td><td>50-230-0316</td><td></td></tr><tr><td>Proteinase K</td><td>5 PRIME</td><td>2500140</td><td></td></tr><tr><td>RNAse A</td><td>5 PRIME</td><td>2500130</td><td></td></tr><tr><td>Salmon Sperm DNA/Protein A Agarose</td><td>EMD Millipore</td><td>16-157</td><td></td></tr><tr><td>Salmon Sperm DNA/Protein G Agaorse</td><td>EMD Millipore</td><td>16-201</td><td></td></tr><tr><td>SDS</td><td>Fisher Scientific</td><td>BP166-500</td><td></td></tr><tr><td>Tris-Cl</td><td>Sigma-Aldrich</td><td>T5941</td><td></td></tr><tr><td>Triton X-100</td><td>Acros Organics</td><td>327372500</td><td></td></tr><tr><td>Anti-K36me3</td><td>Abcam</td><td>ab9050</td><td></td></tr><tr><td>Anti-STAT1</td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-345X</td><td></td></tr><tr><td>Anti-RNA Polymerase II</td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-899</td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

The chip experiment is used to monitor dynamic changes in histo modifications that occur during STAT one activated gene expression alterations in the occupancy of transcription factors, histone modifying complexes and the transcription machinery itself can also be assayed in this way. To begin the assay cells are induced with interferon gamma to activate STAT one signaling cross-linking the cells with formaldehyde and collecting them follows proceed to isolate nucle and sonicate, which shears the chromatin into approximately 200 to 1000 base pair fragments. Next, add an antibody that targets the protein or histo modification of interest and then precipitate the immuno complexes.The immuno complexes are washed, the cross links are reversed and the DNA is purified. The changes in histo modifications or recruitment of proteins to specific areas of a gene can be determined based on quantitative realtime PCR analysis of the immuno precipitated DNA. Hi, my name is Lauren Borow from the laboratory of Dr.Melissa Hendrickson and the Department of Biology at the University of Virginia.Today we're gonna show you a procedure for chromatin immunoprecipitation. We use this procedure in our lab to study dynamic histo modifications that occur during transcription downstream of STAT one signaling. So let's get started.One day before beginning this procedure, culture the cell so that a sufficient number is available for the two FTGH cells used Here. Prepare one 15 centimeter tissue culture dish at 80%co fluency for each chip assay. In each experiment include a negative IgG control, a positive pan histone antibody control and duplicates.To begin this procedure, remove the culture media. Replace it with 20 milliliters of DMEM supplemented with 10%cosmic calf serum and containing interferon gamma at five nanograms per milliliter. Also, replace the media on the un induced plates with 20 milliliters of 10%C-C-S-D-M-E-M.Next work in a fume hood. Tilt the 15 centimeter tissue culture dish and add 540 microliters of a 37%formaldehyde solution to 20 milliliters of media. Then rock the plate to spread it evenly.Allow cross-linking to perceive the 10 minutes at room temperature. Then add glycine to 125 millimolar Final concentration to stop the cross-linking reaction. Vacuum aspirate the media and wash the cells once with 10 milliliters of ice cold PBS using a cell scrapper, collect the cells in around seven milliliters of PBS and combine cells that were treated identically from six 15 centimeter plates in a 50 milliliter conical tube on ice.Finally centrifuge the collected cells at 1, 800 RPM for five minutes at four degrees Celsius and aspirate the PBS. If stopping here, snap, freeze the cell palette and store at minus 80 degrees Celsius after cross-linking. The next step is preparing the chromatin.Start by adding one millimolar DTT and complete protease inhibitors to swelling buffer chilled on ice. Resus suspend the pellet from six plates, pulled in four milliliters of the swelling buffer and transfer to a 15 milliliter conical tube. Using more or less buffer might change the dancing efficiency.Incubate the cell suspension on ice for 10 minutes. When the incubation on ice is complete, transfer the suspension to a pre chilled 15 milliliter downer and dance. 20 strokes with PEs A after dancing transfer the cells back to a 15 milliliter conical tube on ice centrifuge at 2, 500 RPM for five minutes at four degrees Celsius and discard the supernatant leaving the nuclear pellet.Add complete protease inhibitors to sonication buffer chilled on ice. Six plates yield around 200 microliters of nuclear pellet Resus. Suspend the pellet in 15 pellet volumes or three milliliters of the sonication buffer.Using a different volume for this amount of cells might change the sonication efficiency. Sonication efficiency should be determined beforehand for your lab's ator. Here a Miss Sonic sonicate 3000 equipped with the microchip is used.Sonicate the nuclei suspension on ice for 10 cycles for 20 seconds on and 40 seconds off at an amplitude setting of eight. This typically results in chromatin fragments averaging 200 to 1000 base pairs in size when sonication is complete. Transfer the sonicated material to an SS 34 centrifuge tube and centrifuge at 13, 000 RPM for 20 minutes of four degrees Celsius.In an SS 34 rotor to pellet cell debris, which is usually a small amount. Carefully transfer the supinate to a 15 milliliter conical tube on ice. Proceed to chip to decrease background from non-specific binding events.First, pre-clear the chromatin preparation by adding 360 microliters of salmon sperm DNA protein A or G agro bead slurry and rocking for four degrees Celsius for at least 30 minutes. Centrifuge at 1000 RPM for five minutes at four degrees Celsius to pellet the agro beads. Then transfer the pre-cleared supine natin to another 15 milliliter conal tube on ice.Next, measure the absorbance of 260 nanometers or the A 260 units of the chromatin. Dilute 10 microliters of preclude chromatin in 190 microliters of double distilled water. For the blank, combine 10 microliters of sonication buffer with 190 microliters of double distilled water.Adjust the volume of the sample with sonication buffer to four A 260 units. Save 75 microliters of the pre-cleared chromatin at four a two 60 units as the input sample and store at minus 20 degrees Celsius until ready to reverse cross links. Remember that this step is crucial for data analysis.So do not neglect to take the input aliquot dispense the remaining chromatin preparation in one milliliter aliquots into 1.7 milliliter micro refuse tubes on ice. If stopping here, snap. Freeze the prepared chromatin on dry ice and store at minus 80 degrees Celsius.However, since stored chromatin can degrade, it is best to continue with the ip. Now add the chip grade antibodies to the induced and un induced sample sets. In duplicate.Typically one to two micrograms of antibody works well, but the amount must be determined empirically beforehand. Add two micrograms of IgG and 15 microliters of pan histone H three antibody as the negative and positive controls to induced and un induced sample sets. As well rock all tubes overnight at four degrees Celsius before proceeding to isolate the immuno complexes following the overnight incubation with the antibodies.Add 60 microliters of salmon sperm DNA protein A or protein giro bead slurry to all tubes. Rocket four degrees Celsius for at least 60 minutes. To bind the immuno complexes to the beads.Pellet the immuno complexes bound to the agros beads by micro gently at 2000 RPM for three minutes at four degrees Celsius aspirate the supernatant. Next perform a series of buffer washes, starting with a low salt wash followed by a high salt wash. Then lithium chloride wash.And finally, two TE washes to each of the wash buffers. Add PMSF just prior to use wash with one milliliter of buffer for 10 minutes with rocking at four degrees Celsius micro for three minutes at 2000 RPM at four degrees Celsius and aspirate the supernatant keeping the samples chilled on ice. After removing the final tea buffer wash, add 500 microliters of freshly prepared Lucian buffer to the beads vortex to mix the aros beads in the Lucian buffer and heat for 30 minutes at 65 degrees Celsius.Vortexing every 10 minutes while the samples are eluting, thaw the input samples and add 500 microliters of elution buffer to each one. Include these samples in all steps going forward. Once the samples finish the 65 degree Celsius incubation micro fusion per 30 seconds at 2000 RPM and transfer the EENT to a new micro fuge tube.Finally, reverse the cross-linking. Add five molar sodium chloride to a final concentration of 200 millimolar to all samples and vortex incubator 65 degrees Celsius for at least four hours up to overnight. Once the cross links of the immuno complexes are reversed, the bound DNA can be recovered when the incubation at 65 degrees Celsius is complete.Micro fuge samples for 30 seconds. To collect condensation on lids, add one microliter of RNAs A to each tube and incubate room temperature for 15 minutes following the RNAs treatment to each tube at 10 microliters of 0.5 molar EDTA 40 microliters of 0.5 molo tris chloride pH 6.5 and two microliters of proteinase K or a total of 52 microliters from a master mix and incubate at 45 degrees Celsius for 60 minutes. Extract the samples with phenol chloroform, iso aile alcohol, and then with chloroform ISO A my alcohol.Then to precipitate the DNA, add two microliters of glycogen and 0.1 volumes of three molar sodium acetate. pH 5.2 to each sample and vortex, add 2.5 volumes of 100%ethanol vortex and incubate at minus 20 degrees Celsius overnight. Retrieve the samples and micro refuse at 13, 000 RPM for 20 minutes at four degrees Celsius.Wash the DNA glycogen pellets with one milliliter of 70%ethanol and air. Dry the pellets or use a speed vacuum centrifuge. Do not over dry the DNA re suspend the pellets in 200 microliters of TE buffer and quantify no more than one microliter by real time.PCR shown here are the results of a chip experiment following the dynamics of H three K 36 ME three. During interferon gamma induction of the IRF one gene H three K 36 ME three antibody was used to pull down chromatin collected from two FTGH cells induced with interferon gamma for 30 minutes, 1.5 hours and five hours with IgG as a negative control. The occupancy of STAT one was also determined at these time points as was RNA polymerase two occupancy.The histone occupancy across the IRF one gene locus was determined for the un induced and induced conditions using the pan histone three antibody, which also serves as a positive control. The dip around minus five base pairs is due to the nucleosome depletion that is found at transcription start sites. We've just shown you the procedure for chromatin immunoprecipitation.This assay is useful for observing chromatin modifications that occur during activated gene transcription. When doing this procedure, it is important to work out the shearing procedure for your chromatin with your ator before starting a chip experiment. Also remember to take your input samples.So that's it. Thanks for watching and good luck with your experiments.

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Research

JoVE Journal

Biology

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation. <br...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Assays for Validating Histone Acetyltransferase Inhibitors

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).

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Learn chromatin immunoprecipitation to study dynamic histone modifications during STAT1-activated gene expression in human cells. This method enables monitoring of transcription factor occupancy and chromatin changes.

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

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