This work was supported by NIH grants CA141009 and GM39067. We thank E. Parnell and R. Yarrington for comments on the written portion.

1. Isolation of Mouse Splenic Na&iuml;ve CD4 T Cells
<ol>
 <li>Sacrifice the mouse in a humane manner consistent with Institutional Animal Care and Use Committee (IACUC) protocols. Dissect the spleen and place it in Petri dish containing 10 ml of DMEM with 10% FBS.</li>
 <li>Crush the spleen using frosted ends of two glass slides to release the splenocytes. Transfer the cell suspension in a 15 ml conical tube.</li>
 <li>Collect the cells by centrifugation at 200 x g (~1,200 rpm for a clinical centrifuge with a typical rotor diameter) for 5 min at 4 &deg;C.</li>
 <li>Resuspend cells in 2 ml ACK buffer to lyse red blood cells, 1 min at room temperature (RT). Stop the reaction by adding 8 ml of DMEM with FBS.</li>
 <li>Collect the cells by centrifugation at 200 x g for 5 min at 4 &deg;C.</li>
 <li>Resuspend the cells in 5 ml of DMEM containing FBS and pass through a 70 &mu;m mesh filter (BD Falcon, Cat# 352350). Count the cells and proceed to isolation of na&iuml;ve CD4 T cells using a CD4 isolation kit (Miltenyi, Cat# 130-095-248) using the manufacturer's instructions. Collect na&iuml;ve CD4 T cells by centrifugation at 200 x g for 5 min at 4 &deg;C. Resuspend the cells in 10 ml of DMEM with FBS.</li>
</ol>
2. Preparation of Chromatin
<ol>
 <li>Add 37% formaldehyde to a final concentration of 1% to the cell suspension in DMEM and gently rock at RT (e.g. using a Nutator) for 15 min to cross-link DNA:protein complexes.</li>
 <li>Stop crosslinking by adding 1 M glycine to a final concentration of 125 mM. Continue to rock for 5 min at RT.</li>
 <li>Collect the cells by centrifugation at 200 x g for 5 min at 4 &deg;C.</li>
 <li>Wash the cells by resuspending in 5 ml of ice-cold PBS containing protease inhibitors. Wash in ice-cold PBS containing protease inhibitors 3 times total and collect the cells by centrifugation at 4 &deg;C.</li>
 <li>Resuspend the cells in 1 ml ice-cold cell lysis buffer containing protease inhibitors. Incubate on ice for 15 min. The efficiency of cell lysis can be determined by resuspending a small aliquot of cells in 0.4% trypan blue solution (Sigma, Cat# T8154) and observing with a microscope.</li>
 <li>Collect the nuclei by centrifugation at 200 x g (typically ~1,200 rpm) for 5 min at 4 &deg;C. Carefully discard the supernatant.</li>
 <li>Resuspend the nuclei in 500 &mu;l of nuclear lysis buffer containing protease inhibitors. Incubate on ice for 15 min.</li>
 <li>Sonicate the cells using a Misonix Sonicator 3,000, probe size 1.6 mm: Output level 4, 15 sec burst, 4 times on ice. Each sample must be cooled on ice for 1-2 min before sonicating it again to prevent overheating of samples. Overheating can cause the reversal of cross links.</li>
 <li>Centrifuge the sonicated chromatin at 16,000 x g (~13,200 rpm in a microcentrifuge) for 5 min at 4 &deg;C.</li>
 <li>Take a 20 &mu;l aliquot of clear supernatant, add DNA loading dye and check the sonicated DNA by electrophoresis through a 2% agarose gel (ideal size of sonicated DNA for most applications is 200-500 bp).</li>
 <li>Determine DNA concentration using a UV spectrophotometer. Sheared chromatin can be used immediately to set up chromatin immunoprecipitation reaction or stored at -80 &deg;C. Typically we obtain 7.5-10 &mu;g DNA from 2-3 X 106 purified CD4 T cells per mouse.</li>
</ol>
3. Chromatin Immunoprecipitation (All Steps Must be Carried Out at 0-4 &deg;C)
<ol>
 <li>Dilute sonicated chromatin to a DNA concentration of 5-10 &mu;g/ml (total volume= 1 ml) in ChIP dilution buffer with protease inhibitors.</li>
 <li>Save 100 &mu;l (10%) as input. Store on ice.</li>
 <li>Aliquot 450 &mu;l each into two 1.7 ml microfuge tubes labeled as isotype control (mouse or rabbit IgG) and antibody of interest. If multiple antibodies of the same isotype are used, a single isotype control will be sufficient to perform this analysis. You will need multiple isotype controls if antibodies of a different animal source or isotype are used.</li>
 <li>Add 2-5 &mu;g of specific antibody, depending on the specificity of the antibody used, or isotype control to the respective tubes.</li>
 <li>Rock the tubes using a Nutator overnight at 4 &deg;C to allow formation of chromatin-antibody complexes.</li>
 <li>Add 25 &mu;l of protein G magnetic beads (1:1 slurry of suspended beads) to the above mixture and allow to rock for at least 2 hr at 4 &deg;C. Beads from our vendor can be used directly.</li>
 <li>Place the microfuge tubes on a magnetic stand and allow the beads to collect on the magnetized side.</li>
 <li>Carefully remove the solution by aspiration without disturbing the beads.</li>
 <li>Add 1 ml of low salt wash solution and allow to gently rock for 5 min on a Nutator. Collect the beads using a magnetic stand and remove the wash solution. Repeat once.</li>
 <li>Add 1 ml of high salt wash solution and allow to rock for 5 min on a Nutator. Collect the beads using the magnetic stand and remove the wash solution. Repeat once.</li>
 <li>Add 1 ml of lithium chloride wash solution and allow to rock for 5 min on a Nutator. Collect the beads using the magnetic stand and remove the wash solution. Repeat once. The use of LiCl improves the effective removal of non-specific chromatin interactions with the beads.</li>
 <li>Add 1 ml of TE solution and allow to rock for 5 min on a Nutator. Collect the beads using the magnetic stand and remove the wash solution.</li>
 <li>Elute the DNA from the beads by adding 250 &mu;l of elution buffer. Rock for 15 min at room temperature. Pipette off the eluate and save this material in a new 1.7 ml microfuge tube. Repeat once more and combine both the elutions. Discard the beads.</li>
 <li>To reverse the cross-links add 5 M NaCl to a final concentration of 0.3 M and 1 &mu;l of RNase A (20 mg/ml) to the eluted DNA. Add 400 &mu;l of elution buffer to the inputs saved at step 3.2, to make the volume 500 &mu;l. To the inputs add NaCl to 0.3 M, 1 &mu;l of RNase A, 10 &mu;l of 0.5 M EDTA, 20 &mu;l of 1 M Tris-HCl pH 6.5 and 1 &mu;l of proteinase K (20 mg/ml).</li>
 <li>Incubate the tubes overnight at 65 &deg;C in a dry heating block. To avoid evaporation of samples, seal or place a weight on the tubes to keep them from opening. A hybridization oven can also be used.</li>
 <li>Let the tubes cool to RT. Add 1 ml 100% ethanol and incubate 2 hr-overnight at -80 &deg;C to precipitate DNA.</li>
 <li>Centrifuge the tubes at 16,000 x g for 15 min to pellet the DNA. Wash the DNA pellet once with 70% ethanol and air-dry the pellet. Resuspend the DNA pellet in 100 &mu;l of autoclaved distilled water.</li>
 <li>Purify DNA using QiaQuick spin columns and elute in total volume of 50 &mu;l of elution buffer. This DNA is ready for use for PCR.</li>
</ol>
4. Preparation of the PCR Reaction (All Steps Must be Carried Out on Ice)
<ol>
 <li>Collect all the DNA samples from ChIP and corresponding input samples. Also, collect all the PCR reagents and primers for targeted region as well as a control region. Use a "hotstart" Taq DNA polymerase. We will use a SYBR Green-based assay to quantify DNA amplification in this procedure, but qPCR mastermix kits can be used if necessary.</li>
 <li>Make a serial dilution of input DNA to generate a standard curve in the qPCR analysis: for example 10%, 1%, 0.1% and 0.01% in elution buffer (from the QiaQuick spin columns in step 3.18). The concentration range and increment of this standard curve template set can vary based on the expected levels of ChIP enrichment, etc. Dispense 10 &mu;l of input DNA samples in respective wells of a 96-well plate (Genemate, cat# T-3182-1), in duplicate.</li>
 <li>Dispense 10 ml ChIP DNA from isotype control as well as specific antibody in respective wells, in triplicate.</li>
 <li>Make a "master mix" containing either target primers or control primers (0.1 &mu;M final concentration of each primer) and dispense 10 &mu;l into respective wells. For example, in the template below dispense master mix-containing targeted primers into wells marked green and dispense master mix with control primers into wells marked red.</li>
 <li>Cover the PCR plate with optical plastic sealing film and centrifuge at 500 x g in a clinical centrifuge (~1,200 rpm) for 1 min at RT to collect everything at the bottom of the well.</li>
 <li>Start the PCR reaction. We will use the LightCycler 480 II (Roche) operating LightCycler 480SW 1.5 software to run the qPCR in this example.</li>
</ol>
Pre-incubation: 1 cycle: 95 &deg;C/5 min.
Amplification: 40-45 cycles: 94 &deg;C/5 sec, 60 &deg;C/5 sec, 72 &deg;C/10 sec (annealing temperature should be 2-3 &deg;C less than the melting temperature of the primers).
Melting curve: 1 cycle.
Cooling: 1 cycle.
<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td>1</td>
 <td>2</td>
 <td>3</td>
 <td>4</td>
 <td>5</td>
 <td>6</td>
 <td>7</td>
 <td>8</td>
 <td>9</td>
 <td>10</td>
 <td>11</td>
 <td>12</td>
 </tr>
 <tr>
 <td>A</td>
 <td>10% Input</td>
 <td>10% Input</td>
 <td>Isotype</td>
 <td>Specific</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>B</td>
 <td>1% Input</td>
 <td>1% Input</td>
 <td>Isotype</td>
 <td>Specific</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>C</td>
 <td>0.1% Input</td>
 <td>0.1% Input</td>
 <td>Isotype</td>
 <td>Specific</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>D</td>
 <td>0.01% Input</td>
 <td>0.01% Input</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>E</td>
 <td>10% Input</td>
 <td>10% Input</td>
 <td>Isotype</td>
 <td>Specific</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>F</td>
 <td>1% Input</td>
 <td>1% Input</td>
 <td>Isotype</td>
 <td>Specific</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>G</td>
 <td>0.1% Input</td>
 <td>0.1% Input</td>
 <td>Isotype</td>
 <td>Specific</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>H</td>
 <td>0.01% Input</td>
 <td>0.01% Input</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>
Green: Use targeted region primers.
Red: Use control region primers.
5. Analysis
<ol>
 <li>Program the qPCR software for quantification of absolute quantity of DNA. Importantly, the use of a standard curve to interpolate DNA amounts in the unknown specific and isotype samples allows the evaluation and matching of the quality and efficiency of all primer sets over the range of concentrations found in the samples. Furthermore, it also provides a more reliable method to convert the Cq (Ct or Cp) values to final relative fold enrichment results than using&Delta;&Delta;Cq and related methods.</li>
 <li>Go to the sample editor and mark the wells containing input samples of various dilutions (10%, 1%, 0.1% and 0.01%) with their standard curve values. Also label the wells with unknown ChIP samples, exactly as the PCR plate is laid out. In equivalent software used in other qPCR machines, designate subsets corresponding to reactions for each primer pair, standard curve value and experimental and isotype control sample accordingly.</li>
 <li>In the LightCycler software, go to the subset editor and mark the subsets including the wells with input samples as well as the unknown ChIP samples. The unknown samples will be quantified using the standard curve generated from the known concentrations of input samples for the analysis. In equivalent software used in other qPCR machines, designate subsets corresponding to all the reactions for each primer pairs.</li>
 <li>After the PCR amplification is complete, perform the analysis with "Abs Quant/2nd Derivative Max" and select the subset for analysis and press OK. In equivalent software used in other qPCR machines, convert Cq value to the DNA amount in each subset.</li>
 <li>Press "Calculate" which will generate the standard curve using the known concentrations of input samples and will display the absolute quantity of DNA present in the unknown samples.</li>
 <li>If the quality of sheared DNA is good, and if the primers bind specifically to the targeted region, PCR efficiency should be close to 2.</li>
 <li>Perform a melting curve analysis with "Tm Calling" for the same subset, if available. A single peak indicates that only one specific product was amplified. Amplification of specific DNA can also be tested by electrophoresing the PCR product along with DNA ladder through an agarose gel.</li>
 <li>Export data as a tab-delimited text file, which can be opened in Microsoft Excel for further analysis.</li>
 <li>Using Microsoft Excel, divide the DNA amount for the specific antibody with the isotype control for targeted primers. Repeat this step for the control region primers. If multiple antibodies of the same isotype are used for different immunoprecipitations, normalize each of these with the values from the same isotype control. Moreover, if multiple targeted primer pairs are used, the DNA quantities from a single control primer pair set should be used for normalization of each target (Figures 1 and 2). The output values represent the specific antibody immunoprecipitation fold enrichment at each location relative to non-specific antibody background immunoprecipitation.</li>
 <li>Divide the fold enrichment (to isotype control) for targeted primers with the fold enrichment (to isotype control) for control region primers to obtain enrichment relative to a control unbound region (Figure 1). Importantly, note that because of the generation of standard curves with total input DNA for each sample, each of the above immunoprecipitation values interpolated from the standard curves are already normalized to, and expressed as, the fraction of total input by the machine software.</li>
 <li>If the stringency of the buffers used for immunoprecipitation or washing is too high, it is possible that robust amplification from samples immunoprecipitated with specific antibodies will be observed, while amplification from isotype control immunoprecipitated samples will not be observed. In such a scenario, it is better to subtract the amount of Isotype control ChIP from specific antibody ChIP for both the targeted region and the unbound region. Relative enrichment can be calculated by dividing the difference for targeted region by difference for unbound region (Figure 3).</li>
</ol>

<ol>
	<li>Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q., Farnham, 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=Use+of+chromatin+immunoprecipitation+to+clone+novel+E2F+target+promoters.">Use of chromatin immunoprecipitation to clone novel E2F target promoters.</a> Molecular and Cellular Biology. 21, 6820-6832 (2001).</li><li>Weinmann, A. S., Farnham, 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=Identification+of+unknown+target+genes+of+human+transcription+factors+using+chromatin+immunoprecipitation.">Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation.</a> Methods. 26, 37-47 (2002).</li><li>Li, Q., 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=Constitutive+nuclear+localization+of+NFAT+in+Foxp3++regulatory+T+cells+independent+of+calcineurin+activity.">Constitutive nuclear localization of NFAT in Foxp3+ regulatory T cells independent of calcineurin activity.</a> J. Immunol. 188, 4268-4277 (2012).</li><li>Shakya, A., Kang, J., Chumley, J., Williams, M. A., Tantin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oct1+is+a+switchable,+bipotential+stabilizer+of+repressed+and+inducible+transcriptional+states.">Oct1 is a switchable, bipotential stabilizer of repressed and inducible transcriptional states.</a> The Journal of Biological Chemistry. 286, 450-459 (2011).</li><li>Dahl, J. A., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+micro+chromatin+immunoprecipitation+assay+(microChIP).">A rapid micro chromatin immunoprecipitation assay (microChIP).</a> Nature Protocols. 3, 1032-1045 (2008).</li><li>Lubelsky, Y., Macalpine, H. K., Macalpine, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+localization+of+replication+factors.">Genome-wide localization of replication factors.</a> Methods. 57, 187-195 (2012).</li><li>O'Neill, L. P., VerMilyea, M. D., Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+characterization+of+the+early+embryo+with+a+chromatin+immunoprecipitation+protocol+applicable+to+small+cell+populations.">Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations.</a> Nature Genetics. 38, 835-841 (2006).</li><li>Sikes, M. L., 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=A+streamlined+method+for+rapid+and+sensitive+chromatin+immunoprecipitation.">A streamlined method for rapid and sensitive chromatin immunoprecipitation.</a> Journal of Immunological Methods. 344, 58-63 (2009).</li><li>Rhee, H. S., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+genome-wide+protein-DNA+interactions+detected+at+single-nucleotide+resolution.">Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution.</a> Cell. 147, 1408-1419 (2011).</li><li>Rhee, H. S., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+structure+and+organization+of+eukaryotic+pre-initiation+complexes.">Genome-wide structure and organization of eukaryotic pre-initiation complexes.</a> Nature. 483, 295-301 (2012).</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>Robertson, 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=Genome-wide+profiles+of+STAT1+DNA+association+using+chromatin+immunoprecipitation+and+massively+parallel+sequencing.">Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing.</a> Nature Methods. 4, 651-657 (2007).</li></ol>

No conflict of interest declared.

The protocol above provides a robust method of accurately quantifying DNA enrichment from primary lymphocytes using ChIP. One major reason for robustness in this protocol is the inclusion of biological replicates. The above protocol uses three replicates, the enrichment for which is calculated independently. The outputs are then averaged to provide a degree of enrichment and standard deviations calculated to provide a measure of variability. For each sample, three technical replicates are also performed to elimina...

<table><tbody><tr><td>Formaldehyde</td><td>Sigma</td><td>F-8775</td><td>Store at RT</td></tr><tr><td>Phosphate Buffered Saline</td><td>Hyclone</td><td>SH30256.01</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Protease Inhibitor tablets</td><td>Roche</td><td>04693116001</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Protein G magnetic beads</td><td>Active Motif</td><td>101945</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>RNase A (20 mg/ml)</td><td>EMD Millipore</td><td>556746</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Proteinase K (20 mg/ml)</td><td>Roche</td><td>03115879001</td><td>Dissolve in 50 mM Tris-HCl, 10 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, pH 8.0</td></tr><tr><td>Platinum Taq DNA Polymerase</td><td>Invitrogen</td><td>10966-034</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>SYBR Green I</td><td>Invitrogen</td><td>S7567</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>1 M Glycine</td><td></td><td></td><td>Store at RT</td></tr><tr><td>Cell lysis buffer (5 mM Pipes, pH 8.0; 85 mM KCl; 0.5% NP-40)</td><td></td><td></td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Nuclear lysis buffer (50 mM Tris, pH 8.1; 10 mM EDTA; 1% SDS)</td><td></td><td></td><td>Store at RT</td></tr><tr><td>ChIP dilution buffer (0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris pH 8.1; 190 mM NaCl)</td><td></td><td></td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Low salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris pH 8.1; 150 mM NaCl)</td><td></td><td></td><td>Store at 4 &amp;deg;C</td></tr><tr><td>High salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris pH 8.1; 600 mM NaCl)</td><td></td><td></td><td>Store at 4 &amp;deg;C</td></tr><tr><td>LiCl wash buffer (0.25 M LiCl; 1% NP-40; 1% Sodium Deoxycholate, 1 mM EDTA; 10 mM Tris pH 8.0)</td><td></td><td></td><td>Store at 4 &amp;deg;C</td></tr><tr><td>TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA)</td><td></td><td></td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Elution buffer (1% SDS; 0.1 M NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td></td><td></td><td>Prepare fresh</td></tr><tr><td>5 M NaCl</td><td></td><td></td><td>Store at RT</td></tr><tr><td>0.5 M EDTA</td><td></td><td></td><td>Store at RT</td></tr><tr><td>1 M Tris-HCl, pH 6.5</td><td></td><td></td><td>Store at RT</td></tr><tr><td colspan="4">&lt;strong&gt;Table of Specific Reagents&lt;/strong&gt;</td></tr><tr><td>Clay Adams Brand Nutator</td><td>Becton Dickinson</td><td>Model: 421105</td><td></td></tr><tr><td>Magnetic Stand</td><td>Promega</td><td>Z5342</td><td></td></tr><tr><td>Qiaquick PCR Purification Kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>Masonix Sonicator 3000</td><td>QSonica</td><td>Model: S3000</td><td></td></tr><tr><td>UV Spectrophotometer</td><td>NanoDrop Technologies</td><td>ND-1000</td><td></td></tr><tr><td>Heating Block</td><td>VWR</td><td>13259-030</td><td></td></tr><tr><td>Rotator</td><td>VWR</td><td>80085-692</td><td></td></tr><tr><td>Refrigerated bench top centrifuge</td><td>Beckman Coulter</td><td>Model: Allegra X-12R</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415 D</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table of Equipment&lt;/strong&gt; </td></tr></tbody></table>

efficient chromatin immunoprecipitation using limiting amounts of biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

The Chromatin Immunoprecipitation (ChIP) protocol presented here controls for differences, if any, in the amount of DNA used in PCR through use of a primer pair that amplifies an unbound region of genome, thus serving as a "loading control". In the example shown in Figure 3, we have used the coding region of the mouse Actb gene as a region unbound to our protein of interest and the transcription factor NFAT binding site on mouse Il2 promoter as target region. Alternatively, a regio...

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Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

efficient-chromatin-immunoprecipitation-using-limiting-amounts

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JoVE Journal

Biology

14.2K Views.  University of Utah School of Medicine. We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Video: Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Genetics

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

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

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) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

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 ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

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.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

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.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

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 ...

Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells

Source: Fuchs, A. R.,et al. <a href="https://www.jove.com/t/58270">A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia.</a> J. Vis. Exp. (2018).
This video describes protocol for chromatin immunoprecipitation (ChIP) assay, which is a classic technique to demonstrate protein-DNA interactions.This technique is used to identify target genes in human chronic lymphocytic leukemia cells.

Test immunoprecypitacji chromatyny: technika badania interakcji białko-DNA w komórkach

Source: Fuchs, A. R.,et al. A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia. J. Vis. Exp. ...