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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	<li>Rothenberg, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromatin+landscape+and+transcription+factors+in+T+cell+programming.">The chromatin landscape and transcription factors in T cell programming.</a> Trends Immunol. 35 (5), 195-204 (2014).</li><li>Zhang, J. A., Mortazavi, A., Williams, B. A., Wold, B. J., Rothenberg, E. 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. 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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 border="0" cellpadding="0" cellspacing="0" width="912">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:496px;">Agilent High Sensitivity D5000 Reagents</td>
			<td style="width:223px;">Agilent Technologies</td>
			<td style="width:129px;">5067-5593</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Agilent High Sensitivity D5000 ScreenTape</td>
			<td>Agilent Technologies</td>
			<td>5067-5592</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Agilent Tapestation 4200</td>
			<td>Agilent Technologies</td>
			<td>G2991AA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Covaris S220 AFA System</td>
			<td>Covaris</td>
			<td>500217</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">dimethyl adipimidate (DMA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>20660</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine serum (FBS)</td>
			<td>Pan-Biotech</td>
			<td>1502-P121301</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Formaldehyde (FMA)</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Insulin solution human&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>I9278-5ML</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Iscove&#39;s modified Dulbecco&#39;s medium (IMDM)</td>
			<td>Gibco</td>
			<td>21980-032</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Minimum essential medium non-essential amino acids (MEM NEAA)</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">nProtein A Sepharose 4 Fast Flow</td>
			<td>GE Healthcare</td>
			<td>17-5280-02</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Peptone primatone</td>
			<td>Sigma-Aldrich</td>
			<td>P4963-100G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Phase Lock Gel Heavy 2 ml</td>
			<td>5 Prime</td>
			<td>2302830</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Phenol/chloroform/isoamyl alcohol (25:24:1)</td>
			<td>Roth</td>
			<td>A156.2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Phosphate-buffered saline (PBS)</td>
			<td>GIBCO</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Proteinase K Solution (20 mg/mL), RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>25530049</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">QIAquick PCR Purification Kit (250)</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Qubit 3.0 Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q33216</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Qubit assay tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32854</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">RNase A, DNase and protease-free (10 mg/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tube AFA Fiber &#38; 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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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

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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

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

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. ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

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, ...

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

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

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

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 ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

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 ...