We are grateful to P. K&#228;se and T. Schmidt-W&#246;ll for excellent technical assistance. This work was supported by the collaborative research grant TRR81 and the Heisenberg program (BO 1639/5-1) of the DFG (German Research Foundation), the Max Planck Society and the EXC 294 in Freiburg, and the Excellence Cluster for Cardio Pulmonary System (ECCPS) in Giessen to T.B.

NOTE: All the buffers and the medium used in this protocol are listed in Table 1.
Days 1 and 2
1. Crosslinking the Cells
<ol>
	<li>Transfer the mouse T-cells from a 6-well plate to a 50 mL tube and centrifuge for 5 min at 4 &#176;C and ~271 x g. Resuspend the cell pellet in 30 mL of IMDM cell culture medium and count the cells using a Neubauer chamber.</li>
	<li>Collect aliquots of 20 x 106 cells into new 50 mL tubes.</li>
	<li>Add formaldehyde (FMA) to a 1% final concentration directly to the cell culture medium and incubate at room temperature for 10 min. 
	Caution: FMA is toxic if inhaled, so work under a fume hood and wear proper protective equipment. Also, please handle FMA waste according to the rules of the host institution.</li>
	<li>Add a 1/8-volume of 1 M glycine, pH 7.0, and incubate at room temperature for 5 min.</li>
	<li>Pellet the cells by centrifugation for 5 min at 4 &#176;C and ~271 x g and wash with 10 mL of phosphate-buffered saline (PBS).</li>
	<li>Wash the cell pellet with 1 mL of PBS and transfer to 1.5 mL tubes.</li>
</ol>
2. Cell Lysis and Chromatin Shearing
<ol>
	<li>Resuspend the cell pellet in 1 mL of SDS lysis buffer and incubate on ice for 10 min.</li>
	<li>Transfer each sample to a sonication tube and perform shearing using a focused ultrasonicator according to the settings indicated in Table 2.</li>
	<li>After shearing, transfer the samples to 1.5 mL tubes and centrifuge for 10 min at 4 &#176;C and ~18,000 x g.</li>
	<li>Transfer the supernatant to new tubes.</li>
	<li>Collect 50 &#181;L to determine the shearing efficiency according to step 3 and snap-freeze the sheared lysate in liquid nitrogen. Store the lysate in single-use aliquots at -80 &#176;C.</li>
</ol>
3. Determination of Shearing Efficiency
<ol>
	<li>Add 50 &#181;L of elution buffer to the 50 &#181;L of each sheared lysate and incubate at 65 &#176;C overnight, with shaking.</li>
	<li>Add 100 &#181;L of TE buffer and 4 &#181;L of 10 mg/mL RNase A (0.2 &#181;g/&#181;L final concentration). Mix and incubate for 2 h at 37 &#176;C, with shaking.</li>
	<li>Add 2 &#181;L of 20 mg/mL proteinase K (0.2 &#181;g/&#181;L final concentration) to each sample and incubate for 2 h at 55 &#176;C, with shaking.</li>
	<li>Transfer each sample to a gel tube suitable for phenol/chloroform/isoamyl alcohol extraction; handle according to the manufacturer&#39;s instructions.</li>
	<li>Add 200 &#181;L of phenol/chloroform/isoamyl alcohol (25:24:1) and shake the tubes. Centrifuge for 5 min at 24 &#176;C and ~16,000 x g and transfer the upper phase to new tubes. 
	Caution: Phenol, chloroform, and isoamyl alcohol are toxic if inhaled and are corrosive; work under a fume hood and wear proper personal protective equipment. Also, handle phenol, chloroform, and isoamyl alcohol waste according to the rules of the host institution.
	<ol>
		<li>Repeat once.</li>
	</ol>
	</li>
	<li>Purify the DNA by using purification columns according to the manufacturer&#39;s instruction, with the following modifications:
	<ol>
		<li>Wash the membrane twice. After the last wash, leave the tube open for 2 min at room temperature to evaporate the residual ethanol.</li>
		<li>For the elution, add 50 &#181;L of H2O, incubate at room temperature for 1 min, spin the tube for 1 s to properly wet the membrane, and incubate at room temperature for 1 min before eluting the DNA by centrifugation for 1 min at 24 &#176;C and ~17,900 x g.</li>
	</ol>
	</li>
	<li>Quantify the purified DNA on a fluorometer using a high-sensitivity kit according to the manufacturer&#39;s instructions.</li>
	<li>Analyze approximately 500 ng of the purified DNA on a 1.8% agarose gel and approximately 1 ng on an electrophoresis system using a high-sensitivity kit. 
	NOTE: The expected size of the DNA fragments is between 200 and 500 bp.</li>
</ol>
Days 3 and 4
4. Immunoprecipitation
<ol>
	<li>Dilute 1 volume of sheared lysate with 5 volumes of dilution buffer.</li>
	<li>Preclear with 30 &#181;L/mL protein A agarose beads (prepared according to Appendix A) for 30 min while rotating in the cold room.</li>
	<li>Centrifuge at 4 &#176;C for 5 min at ~750 x g.</li>
	<li>Collect 10% of the input and store it at 4 &#176;C.</li>
	<li>Aliquot the desired amount of lysate into new tubes (see Table 3 for details). Add the desired antibodies to each tube (see Table 3 for details) and rotate overnight at 4 &#176;C.</li>
	<li>Add 40 &#181;L of protein A beads and incubate for 1 h at 4 &#176;C while rotating.</li>
	<li>Wash the beads with 1 mL of low-salt buffer, high-salt buffer, LiCl buffer, and TE buffer (for each wash, incubate for 5 min at 4 &#176;C while rotating and centrifuge for 3 min at 4 &#176;C and ~950 x g). See Table 3 for details.</li>
	<li>Add 110 &#181;L of elution buffer and incubate for 15 min at room temperature, vortexing every 2 min.</li>
	<li>Centrifuge for 3 min at 4 &#176;C and ~950 x g and transfer 100 &#181;L of supernatant to a new 1.5-mL tube.</li>
	<li>Repeat steps 4.7-4.8 with 100 &#181;L of elution buffer and combine the eluates. Add elution buffer up to 200 &#181;L to input the samples.</li>
	<li>Incubate all samples overnight at 65 &#176;C, with shaking.</li>
</ol>
Day 5
5. DNA Purification
<ol>
	<li>Add 200 &#181;L of TE buffer to each eluate and 8 &#181;L of 10 mg/mL RNase A (0.2 &#181;g/&#181;L final concentration). Mix and incubate for 2 h at 37 &#176;C, with shaking.</li>
	<li>Add 4 &#181;L of 20 mg/mL proteinase K (0.2 &#181;g/&#181;L final concentration) to each eluate and incubate for 2 h at 55 &#176;C, with shaking.</li>
	<li>Transfer each sample to a gel tube suitable for phenol/chloroform/isoamyl alcohol extraction; handle according to the manufacturer&#39;s instructions.</li>
	<li>Add 400 &#181;L of phenol/chloroform/isoamyl alcohol (25:24:1) and shake the tubes. Centrifuge for 5 min at 24 &#176;C and ~16,000 x g. Transfer the upper phase to new tubes. 
	Caution: Phenol, chloroform, and isoamyl alcohol are toxic if inhaled and are corrosive; work under a fume hood and wear proper personal protective equipment. Also, handle phenol, chloroform, and isoamyl alcohol waste according to the rules of the host institution.
	<ol>
		<li>Repeat once.</li>
	</ol>
	</li>
	<li>Purify the DNA using purification columns according to the manufacturer&#39;s instructions, with the following modifications:</li>
	<li>Wash the membrane twice. After the last wash, leave the tubes open for 2 min at room temperature to evaporate the residual ethanol.</li>
	<li>For the elution, add 50 &#181;L of H2O, incubate at room temperature for 1 min, spin the tubes for 1 s to properly wet the membrane, and incubate at room temperature for 1 min before eluting the DNA by centrifugation for 1 min at 24 &#176;C and ~17,900 x g.</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+transformations+of+genome-wide+epigenetic+marking+and+transcriptional+control+establish+T+cell+identity.">Dynamic transformations of genome-wide epigenetic marking and transcriptional control establish T cell identity.</a> Cell. 149 (2), 467-482 (2012).</li><li>Cauchy, P., 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=Dynamic+recruitment+of+Ets1+to+both+nucleosome-occupied+and+-depleted+enhancer+regions+mediates+a+transcriptional+program+switch+during+early+T-cell+differentiation.">Dynamic recruitment of Ets1 to both nucleosome-occupied and -depleted enhancer regions mediates a transcriptional program switch during early T-cell differentiation.</a> Nucl Acids Res. 44 (8), 3567-3585 (2016).</li><li>Koch, 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=Transcription+initiation+platforms+and+GTF+recruitment+at+tissue-specific+enhancers+and+promoters.">Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters.</a> Nat Struct Mol Biol. 18 (8), 956-963 (2011).</li><li>Pekowska, A., 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=H3K4+tri-methylation+provides+an+epigenetic+signature+of+active+enhancers.">H3K4 tri-methylation provides an epigenetic signature of active enhancers.</a> EMBO J. 30 (20), 4198-4210 (2011).</li><li>Vanhille, 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=High-throughput+and+quantitative+assessment+of+enhancer+activity+in+mammals+by+CapStarr-seq.">High-throughput and quantitative assessment of enhancer activity in mammals by CapStarr-seq.</a> Nat Commun. 6, 6905 (2015).</li><li>Barski, A., 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=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129 (4), 823-837 (2007).</li><li>Heintzman, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+modifications+at+human+enhancers+reflect+global+cell-type-specific+gene+expression.">Histone modifications at human enhancers reflect global cell-type-specific gene expression.</a> Nature. 459 (7243), 108-112 (2009).</li><li>Ernst, 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=Mapping+and+analysis+of+chromatin+state+dynamics+in+nine+human+cell+types.">Mapping and analysis of chromatin state dynamics in nine human cell types.</a> Nature. 473 (7345), 43-49 (2011).</li><li>Ostuni, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latent+enhancers+activated+by+stimulation+in+differentiated+cells.">Latent enhancers activated by stimulation in differentiated cells.</a> Cell. 152 (1-2), 157-171 (2013).</li><li>Giaimo, B. D., Oswald, F., Borggrefe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+chromatin+regulation+at+Notch+target+genes.">Dynamic chromatin regulation at Notch target genes.</a> Transcription. 8 (1), 61-66 (2017).</li><li>Liefke, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+demethylase+KDM5A+is+an+integral+part+of+the+core+Notch-RBP-J+repressor+complex.">Histone demethylase KDM5A is an integral part of the core Notch-RBP-J repressor complex.</a> Genes Dev. 24 (6), 590-601 (2010).</li><li>Jung, C., Mittler, G., Oswald, F., Borggrefe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+helicase+Ddx5+and+the+noncoding+RNA+SRA+act+as+coactivators+in+the+Notch+signaling+pathway.">RNA helicase Ddx5 and the noncoding RNA SRA act as coactivators in the Notch signaling pathway.</a> Biochim Biophys Acta. 1833 (5), 1180-1189 (2013).</li><li>Oswald, 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=A+phospho-dependent+mechanism+involving+NCoR+and+KMT2D+controls+a+permissive+chromatin+state+at+Notch+target+genes.">A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes.</a> Nucl Acids Res. 44 (10), 4703-4720 (2016).</li><li>Arrigoni, 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=Standardizing+chromatin+research:+a+simple+and+universal+method+for+ChIP-seq.">Standardizing chromatin research: a simple and universal method for ChIP-seq.</a> Nucl Acids Res. 44 (7), e67 (2016).</li><li>Essen, D., Dullforce, P., Brocker, T., Gray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+interactions+involved+in+Th+cell+memory.">Cellular interactions involved in Th cell memory.</a> J Immunol. 165 (7), 3640-3646 (2000).</li><li>White, 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=Two+better+cell+lines+for+making+hybridomas+expressing+specific+T+cell+receptors.">Two better cell lines for making hybridomas expressing specific T cell receptors.</a> J Immunol. 143 (6), 1822-1825 (1989).</li><li>Ghisletti, S., 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=Identification+and+characterization+of+enhancers+controlling+the+inflammatory+gene+expression+program+in+macrophages.">Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages.</a> Immunity. 32 (3), 317-328 (2010).</li><li>Egelhofer, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assessment+of+histone-modification+antibody+quality.">An assessment of histone-modification antibody quality.</a> Nat Struct Mol Biol. 18 (1), 91-93 (2011).</li><li>Rothbart, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Interactive+Database+for+the+Assessment+of+Histone+Antibody+Specificity.">An Interactive Database for the Assessment of Histone Antibody Specificity.</a> Mol Cell. 59 (3), 502-511 (2015).</li><li>Fenouil, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG+islands+and+GC+content+dictate+nucleosome+depletion+in+a+transcription-independent+manner+at+mammalian+promoters.">CpG islands and GC content dictate nucleosome depletion in a transcription-independent manner at mammalian promoters.</a> Genome Res. 22 (12), 2399-2408 (2012).</li><li>Hein, K., 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=Site-specific+methylation+of+Notch1+controls+the+amplitude+and+duration+of+the+Notch1+response.">Site-specific methylation of Notch1 controls the amplitude and duration of the Notch1 response.</a> Sci Signal. 8 (369), ra30 (2015).</li></ol>

The authors have no competing financial interests.

ChIP is a valid technique to investigate whether proteins or their PTMs are enriched at specific genomic regions. ChIP assay results are often challenging to interpret due to biological or technical reasons. The biological reasons are manyfold and include weak or indirect binding of proteins to DNA. There are also technical limitations, such as limited antibody specificity and inefficient cell lysis or chromatin shearing. A troubleshooting guide (Table 5) can help the reader to solve the problems that may be encountered with ChIP assays.
This protocol, making use of a cooling focused ultrasonicator device, allows one to efficiently shear the chromatin of a mature mouse T-cell line. The main critical step of the protocol is represented by the shearing of the chromatin, which must always be optimized for each cell type. It is suggested to vary the number of cells and the number of sonication cycles. Furthermore, the shearing efficiency is negatively influenced by the presence of SDS precipitates in the samples, which may form during the lysis of the cells on ice with SDS lysis buffer. In this case, it is best to incubate the sample after lysis at room temperature for a few minutes to reduce the presence of SDS precipitates. It is recommended to optimize the protocol to obtain sheared fragments between 200 and 500 bp and to always evaluate the shearing quality of the samples before proceeding with the immunoprecipitation. Additionally, the fixation time, amount of antibody and/or cells, and the washing conditions must be optimized for each antibody.
One more critical step in making a ChIP procedure successful is the choice of the antibody, as low-specificity antibodies significantly reduce the efficiency of the immunoprecipitation. Previously, a unified quality test procedure allowed for the evaluation of the specificity of several antibodies available on the market19. The screening pipeline was based on dot blot, Western blot, and ChIP, providing a list of high-quality reagents suitable for ChIP19. More recently, the specificity of several commercially available antibody was evaluated using high-density histone peptide microarray platforms, allowing for the establishment of a database on antibody specificity20. The reader can use this useful tool to identify the most specific antibodies that can be used for ChIP experiments.
This protocol has not been tested for primary T-cells and, in this case, the reader should refer to other published protocols that successfully perform ChIP on primary T-cells3,4,21. Notably, this protocol has been successfully used to perform ChIP on a mouse progenitor T-cell line, as well as on a mouse endothelial cell line.
5 x 106 cells should be used for each immunoprecipitation for the analysis of histone marks. Because this protocol can also be suitable, with some optimization, to perform ChIP on TFs or cofactors, it is best to increase the number of cells used for the immunoprecipitation in these cases. In order to reach the required number of cells for each experiment, more aliquots of sheared lysate can be pooled together before diluting the chromatin in the dilution buffer.
This protocol could also be modified to perform ChIP on endogenous proteins that do not bind directly to the DNA. In this case, pre-fixation with protein-protein crosslinkers (e.g., dimethyl adipimidate, DMA) may be required (see Appendix B for more information). The successful performance of ChIP on cofactors was previously done by overexpressing proteins that are fused to a biotin-tag. In this case, the protein was purified with streptavidin-conjugated magnetic beads, and a pre-fixation with DMA was performed22.

Development, differentiation, and homeostasis depend upon specific gene expression programs that are established by signaling events that modulate chromatin structure and therefore determine if a specific gene is activated or repressed in a cell- and time-specific manner. During T-cell development, specific gene expression programs must be established to properly determine the maturation of T-cell precursors from the double-negative (DN) to the single-positive (SP) state, passing through several intermediate stages1. How the gene expression program is dynamically regulated during T-cell development has been broadly investigated in previous years by several laboratories2,3,4,5,6.
By post-translational modifications (PTMs) of histones, the exchange of canonical histones with histone variants, nucleosome eviction, and DNA methylation regulate the ON/OFF switch of genes. Several groups have investigated the genome-wide distribution of PTMs and histone variants to determine marks that are associated with different chromatin states at both proximal and distal regulatory regions7,8,9,10. Signaling cascades orchestrate dynamic chromatin regulation via the exchange of positive and negative chromatin-modifying enzymes (also known as chromatin modifiers) at specific enhancer elements. These chromatin modifiers regulate the chromatin structure and thus the transcriptional output by, for example, dynamic histone methylation and acetylation. This is the case in the Notch signaling pathway11,12,13,14.
Dynamic histone PTMs; exchanges with histone variants; and the dynamic occupancy of histones, transcription factors, and cofactors can be investigated by chromatin immunoprecipitation (ChIP) assays. Highly specific antibodies are used to purify specific DNA-protein complexes, and the purified DNA is analyzed by quantitative PCR (qPCR); deep-sequencing (ChIP-Seq); or, less frequently nowadays, hybridization to microarray (ChIP-ChIP).
ChIP assays are sometimes challenging due to complications with the lysis of the cells, shearing of the chromatin, and/or low specificity of the antibodies. Several strategies have been adopted to improve the protocol, as is the case for NEXSON15. The use of cooling water-bath sonicators avoids sample heating that might damage the epitopes present on the protein under investigation, but the energy required for the shearing of the samples gets dispersed into the water. Another improvement was made with the development of focused ultrasonication devices that prevent the dispersion of the energy. Therefore, focused ultrasonicator devices allow for improvements in cell lysis and chromatin shearing, eliminating operator-induced variations and significantly increasing the reproducibility.
This work describes a protocol (schematic overview in Figure 1) to efficiently perform the ChIP of histone proteins in a mature mouse T-cell line called E2-10HA16,17. T-cells are usually difficult to lyse, and the shearing of their chromatin has been revealed to be inefficient. In this protocol, which makes use of a cooling focused ultrasonicator, the number of cells, the lysis buffer, and the shearing setting were optimized for the E2-10HA mouse T-cell line. This protocol allows one to perform ChIP with high reproducibility and in a reasonable time. In fact, it requires approximately two days to shear the chromatin and to evaluate the quality of the shearing, and three days to perform the immunoprecipitation, reverse the crosslink, and purify the DNA.

<table><tbody><tr><td>Agilent High Sensitivity D5000 Reagents</td><td>Agilent Technologies</td><td>5067-5593</td><td></td></tr><tr><td>Agilent High Sensitivity D5000 ScreenTape</td><td>Agilent Technologies</td><td>5067-5592</td><td></td></tr><tr><td>Agilent Tapestation 4200</td><td>Agilent Technologies</td><td>G2991AA</td><td></td></tr><tr><td>Covaris S220 AFA System</td><td>Covaris</td><td>500217</td><td></td></tr><tr><td>dimethyl adipimidate (DMA)</td><td>Thermo Fisher Scientific</td><td>20660</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Pan-Biotech</td><td>1502-P121301</td><td></td></tr><tr><td>Formaldehyde (FMA)</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Insulin solution human&amp;nbsp;</td><td>Sigma-Aldrich</td><td>I9278-5ML</td><td></td></tr><tr><td>Iscove&#039;s modified Dulbecco&#039;s medium (IMDM)</td><td>Gibco</td><td>21980-032</td><td></td></tr><tr><td>Minimum essential medium non-essential amino acids (MEM NEAA)</td><td>Gibco</td><td>11140-035</td><td></td></tr><tr><td>nProtein A Sepharose 4 Fast Flow</td><td>GE Healthcare</td><td>17-5280-02</td><td></td></tr><tr><td>Penicillin-Streptomycin&lt;br /&gt;(10,000 U/mL)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Peptone primatone</td><td>Sigma-Aldrich</td><td>P4963-100G</td><td></td></tr><tr><td>Phase Lock Gel Heavy 2&amp;nbsp;mL</td><td>5 Prime</td><td>2302830</td><td></td></tr><tr><td>Phenol/chloroform/isoamyl alcohol (25:24:1)</td><td>Roth</td><td>A156.2</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>GIBCO</td><td>14190-094</td><td></td></tr><tr><td>Proteinase K Solution (20 mg/mL), RNA grade</td><td>Thermo Fisher Scientific</td><td>25530049</td><td></td></tr><tr><td>QIAquick PCR Purification Kit (250)</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33216</td><td></td></tr><tr><td>Qubit assay tubes</td><td>Thermo Fisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Q32854</td><td></td></tr><tr><td>RNase A, DNase and protease-free (10&amp;nbsp;mg/mL)</td><td>Thermo Fisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>Tube AFA Fiber &amp;amp; Cap</td><td>Covaris</td><td>520081</td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) in mouse t-cell lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Mature murine T-cells were subjected to ChIP analysis to investigate the enrichment of the histone mark mono-methylation of lysine 4 on histone 3 (H3K4me1), tri-methylation of lysine 4 on histone 3 (H3K4me3), and acetylation of lysine 27 on histone 3 (H3K27ac), as well as the nucleosome occupancy, as revealed by panH3 ChIP. The shearing quality was evaluated by analysis on a 1.8% agarose gel (Figure 2A) and on an electrophoresis system (Figure 2B). H3K4me1 and H3K4me3 are generally used to identify enhancer sites and promoters, respectively (Figure 3A). In fact, H3K4me3 is prominent but not exclusively enriched at promoters5,8,14. A characteristic of active enhancers is to be highly enriched for H3K4me1 and H3K27ac and poorly enriched for H3K4me3 (Figure 3A, upper panel), whereas inactive enhancers present low levels of H3K4me3 and H3K27ac but high levels of H3K4me1 (Figure 3A, lower panel). As shown in Figure 3B, the Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) gene is representative of the analysis of active gene promoters (Gapdh promoter). In fact, it shows high levels of H3K4me3 and H3K27ac and low levels of H3K4me1. In Figure 3B, Deltex1 (Dtx1) is representative of inactive enhancers (Dtx1 enhancer), as it is highly enriched for H3K4me1 and poorly enriched for H3K4me3 and H3K27ac. Gene desert is representative of a region lacking coding genes and is usually sued as a negative control for active chromatin marks. The observation that H3K4me1, a well-accepted enhancer mark4,5,7,14,18, is not enriched at Gene desert suggests that this region lacks enhancers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55907/55907fig1.jpg" /> 
Figure 1: Schematic overview of the ChIP protocol presented. <a href="http://cloudflare.jove.com/files/ftp_upload/55907/55907fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55907/55907fig2.jpg" /> 
Figure 2: Shearing quality control of the mature mouse T-cell line. (A) Two different aliquots of mature mouse T-cell line E2-10HA were sheared, and approximately 500 ng of purified DNA were analyzed on a 1.8% agarose gel to evaluate their shearing quality. (B) 1 ng of purified DNA from sample 2 was analyzed by electrophoresis to evaluate its shearing quality. <a href="http://cloudflare.jove.com/files/ftp_upload/55907/55907fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55907/55907fig3.jpg" /> 
Figure 3: Chromatin marks that characterize enhancers and promoters. (A) Active enhancers (upper panel) are characterized by high H3K4me1, low H3K4me3, and high H3K27ac, whereas inactive enhancers (lower panel) present high H3K4me1, low H3K4me3, and low H3K27ac. (B) The samples presented in Figure 2A were pooled together and used for ChIP analysis versus histone marks H3K4me1, H3K4me3, and H3K27ac and histone occupancy, as revealed by panH3 ChIP. This analysis shows that the promoter of the housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (Gapdh promoter) is highly enriched for H3K4me3 and H3K27ac and lowly enriched for H3K4me1. On the other hand, the enhancer of the inactive gene Deltex1 (Dtx1 enhancer) is highly enriched for H3K4me1 but poorly enriched for H3K4me3 and H3K27ac. ChIP using a panH3 antibody was used to investigate nucleosome occupancy. Gene desert was used as a negative control. One representative experiment is shown. <a href="http://cloudflare.jove.com/files/ftp_upload/55907/55907fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of the buffer</td>
			<td>Reagent</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td rowspan="5">Dilution buffer</td>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>0.01% (w/v)</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>1.1% (v/v)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA) pH 8.0</td>
			<td>1.2 mM</td>
		</tr>
		<tr>
			<td>Tris-HCl pH 8.1</td>
			<td>16.7 mM</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>167 mM</td>
		</tr>
		<tr>
			<td rowspan="2">DMA solution</td>
			<td>Dimethyl adipimidate (DMA)</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>1x</td>
		</tr>
		<tr>
			<td rowspan="3">Elution buffer</td>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>1% (w/v)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA) pH 8.0</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Tris-HCl pH 8.0</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td rowspan="5">High salt buffer</td>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>0.1% (w/v)</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>1% (v/v)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA) pH 8.0</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Tris-HCl pH 8.1</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>500 mM</td>
		</tr>
		<tr>
			<td rowspan="6">IMDM medium</td>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM)</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>2% (v/v)</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Peptone primatone</td>
			<td>0.3 mg/mL</td>
		</tr>
		<tr>
			<td>Insulin solution human&#160;</td>
			<td>4.8 mg/mL</td>
		</tr>
		<tr>
			<td>Minimum essential medium non-essential amino acids (MEM NEAA)</td>
			<td>1x</td>
		</tr>
		<tr>
			<td rowspan="4">LiCl salt buffer</td>
			<td>Lithium chloride (LiCl)</td>
			<td>0.25 M</td>
		</tr>
		<tr>
			<td>IGEPAL-CA630</td>
			<td>1% (v/v)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA) pH 8.0</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Tris-HCl pH 8.1</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td rowspan="5">Low salt buffer</td>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>0.1% (w/v)</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>1% (v/v)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA) pH 8.0</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Tris-HCl pH 8.1</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td rowspan="5">Protein A Sepharose beads washing buffer</td>
			<td>Tris-HCl pH 8.0</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>500 mM</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA) pH 8.0</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>0.1% (w/v)</td>
		</tr>
		<tr>
			<td>IGEPAL-CA630</td>
			<td>1% (v/v)</td>
		</tr>
		<tr>
			<td rowspan="3">SDS Lysis buffer</td>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>1% (w/v)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA) pH 8.0</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Tris-HCl pH 8.1</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td rowspan="2">TE buffer</td>
			<td>Tris-HCl pH 8.0</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA) pH 8.0</td>
			<td>1 mM</td>
		</tr>
	</tbody>
</table>
Table 1: List of the buffers and the medium used in this protocol.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>ON</td>
			<td>OFF</td>
		</tr>
		<tr>
			<td>Peak power</td>
			<td>150.0</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>Duty factor</td>
			<td>15.0</td>
			<td>15.0</td>
		</tr>
		<tr>
			<td>Cycles/burst</td>
			<td>500</td>
			<td>500</td>
		</tr>
		<tr>
			<td>No. of cycles</td>
			<td colspan="2">28</td>
		</tr>
	</tbody>
</table>
Table 2: Shearing settings used in this protocol. These conditions have been optimized for a mature mouse T-cell line.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Supplier</td>
			<td>Amount of antibody/immunoprecipitation</td>
			<td>Amount of cells/immunoprecipitation</td>
			<td>Washing conditions</td>
		</tr>
		<tr>
			<td>H3</td>
			<td>Abcam (ab1791)</td>
			<td>2.5 mg</td>
			<td>5 x 106</td>
			<td>Once in low salt buffer, twice in High salt buffer, twice in LiCl salt buffer and three times in TE buffer</td>
		</tr>
		<tr>
			<td>H3K4me1</td>
			<td>Abcam (ab8895)</td>
			<td>2.5 mg</td>
			<td>5 x 106</td>
			<td>Once in low salt buffer, twice in High salt buffer, twice in LiCl salt buffer and three times in TE buffer</td>
		</tr>
		<tr>
			<td>H3K4me3</td>
			<td>Diagenode (pAb-003-050)</td>
			<td>2.5 mg</td>
			<td>5 x 106</td>
			<td>Once in low salt buffer, twice in High salt buffer, twice in LiCl salt buffer and three times in TE buffer</td>
		</tr>
		<tr>
			<td>H3K27ac</td>
			<td>Diagenode (pAb-174-050)</td>
			<td>2.5 mg</td>
			<td>5 x 106</td>
			<td>Oce in low salt buffer, twice in High salt buffer, twice in LiCl salt buffer and three times in TE buffer</td>
		</tr>
		<tr>
			<td>IgG</td>
			<td>Diagenode (C15410206)</td>
			<td>Variable*</td>
			<td>Variable*</td>
			<td>Variable*</td>
		</tr>
		<tr>
			<td colspan="5">* In the case of the IgG control, the amount of both antibody and cells, as well as the washing steps, have to mirror the conditions of the other immunoprecipitations.</td>
		</tr>
	</tbody>
</table>
Table 3: Antibodies and washing conditions used in this study.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Gene</td>
			<td>forward primer</td>
			<td>reverse primer</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>Gapdh promoter (0 kb)</td>
			<td>5&#39;-GGG TTC CTA TAA ATA CGG ACT GC-3&#39;</td>
			<td>5&#39;-CTG GCA CTG CAC AAG AAG A-3&#39;</td>
			<td>68</td>
		</tr>
		<tr>
			<td>Dtx1 enhancer (+26 kb)</td>
			<td>5&#39;-CTC TGG GTT GTA GGG GAC AG-3&#39;</td>
			<td>5&#39;-GCA TGG GAA CTG TGT TAC AGA A-3&#39;</td>
			<td>27</td>
		</tr>
		<tr>
			<td>Gene desert</td>
			<td>5&#39;-CAA TGC ATG GGT CCA GAT TT-3&#39;</td>
			<td>5&#39;-ATT GGC ACG GAA GTA GTG CT-3&#39;</td>
			<td>94</td>
		</tr>
	</tbody>
</table>
Table 4: Primers and probes used for qPCR in this study.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Causes</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td rowspan="3">Chromatin is under sheared and fragments are too big.</td>
			<td>Too many cells have been used.</td>
			<td>Reduce the number of cells.</td>
		</tr>
		<tr>
			<td rowspan="2">The shearing is too low.</td>
			<td>Increase the number of shearing cycles.</td>
		</tr>
		<tr>
			<td>Increase the shearing peak power, the duty factor and/or the cycles/burst.</td>
		</tr>
		<tr>
			<td rowspan="3">Chromatin is over sheared and fragments are too small.</td>
			<td>Too few cells have been used.</td>
			<td>Increase the number of cells.</td>
		</tr>
		<tr>
			<td rowspan="2">The shearing is too high.</td>
			<td>Reduce the number of shearing cycles.</td>
		</tr>
		<tr>
			<td>Reduce the shearing peak power, the duty factor and/or the cycles/burst.</td>
		</tr>
		<tr>
			<td rowspan="11">Too low recovery over IgG control and/or negative control (high background).</td>
			<td>Not enough chromatin used for the experiment.</td>
			<td>Increase the amount of chromatin.</td>
		</tr>
		<tr>
			<td>Amount of antibody is too low.</td>
			<td>Increase the amount of antibody.</td>
		</tr>
		<tr>
			<td>Amount of antibody is too high.</td>
			<td>Reduce the amount of antibody.</td>
		</tr>
		<tr>
			<td>The antibody has low specificity.</td>
			<td>Change the antibody.</td>
		</tr>
		<tr>
			<td rowspan="2">Washing conditions are too stringent.</td>
			<td>Reduce the number of washing.</td>
		</tr>
		<tr>
			<td>Reduce the stringency of the washing buffers.</td>
		</tr>
		<tr>
			<td rowspan="2">Washing conditions are not stringent enough.</td>
			<td>Increase the number of washing.</td>
		</tr>
		<tr>
			<td>Increase the stringency of the washing buffers.</td>
		</tr>
		<tr>
			<td>Too much DNA was pipetted in the qPCR.</td>
			<td>Reduce the amount of DNA pipetted in the qPCR.</td>
		</tr>
		<tr>
			<td rowspan="2">qPCR conditions are not optimal.</td>
			<td>Optimize the qPCR conditions.</td>
		</tr>
		<tr>
			<td>Reduce the number of cycles.</td>
		</tr>
		<tr>
			<td rowspan="4">No product or product is too low in the qPCR reaction of the input sample.</td>
			<td>Not enough DNA was pipetted in the qPCR.</td>
			<td>Increase the amount of DNA pipetted in the qPCR.</td>
		</tr>
		<tr>
			<td rowspan="2">qPCR conditions are not optimal.</td>
			<td>Optimize the qPCR conditions.</td>
		</tr>
		<tr>
			<td>Increase the number of cycles.</td>
		</tr>
		<tr>
			<td>Not enough chromatin used for the experiment.</td>
			<td>Increase the amount of chromatin.</td>
		</tr>
		<tr>
			<td rowspan="4">No signal in the positive control and/or panH3 ChIP.</td>
			<td>Not enough chromatin used for the experiment.</td>
			<td>Increase the amount of chromatin.</td>
		</tr>
		<tr>
			<td>Amount of antibody is too low.</td>
			<td>Increase the amount of antibody.</td>
		</tr>
		<tr>
			<td>The antibody has low specificity.</td>
			<td>Change the antibody.</td>
		</tr>
		<tr>
			<td>Elution is sub-optimal.</td>
			<td>Be sure to frequently vortex the samples to maintain the beads in solution.</td>
		</tr>
	</tbody>
</table>
Table 5: Troubleshooting.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines at JoVE.com

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

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

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

chromatin-immunoprecipitation-chip-in-mouse-t-cell-lines

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

18.2K Views.  University of Giessen. 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.

Video: Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

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 ...

Biochemistry

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 from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

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.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Biology

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 ...

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

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 ...

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.

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 ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

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.

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

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

Histones are proteins that help organize DNA in eukaryotic nuclei by serving as &#8220;scaffolds&#8221; around which DNA can be wrapped, forming a complex called &#8220;chromatin&#8221;. These proteins can be modified through the addition of chemical groups, and these changes affect gene expression. Researchers use a technique called chromatin immunoprecipitation (ChIP) to better understand which DNA regions associate with specific histone modifications or other gene regulatory proteins. Antibodies are used to isolate the protein of interest, and the bound DNA is extracted for analysis. Here, JoVE presents the principles behind ChIP, discussing specific histone modifications and their relationship to gene expression and DNA organization. We then review how to perform a ChIP protocol, and explore the ways scientists are currently using this technique.

Histones are proteins that help organize DNA in eukaryotic nuclei by serving as &#8220;scaffolds&#8221; around which DNA can be wrapped, forming a complex ...