The authors would like to thank members of the Green lab for helpful discussions. This work was supported in part by NIH grants R03AG052018 and R01GM124342 to E.M.G.

1. Pre-bind Antibody to Magnetic Beads
<ol>
	<li>Mix the protein A/G magnetic beads well and transfer 20 &#181;L of magnetic beads per one immunoprecipitation (IP) sample to a 1.5 mL tube. 
	NOTE: When pipetting beads, use wide-bore, low-retention tips.</li>
	<li>Place the tube with beads into a magnetic stand and allow beads to collect on side of the tube. Remove the supernatant.</li>
	<li>Wash the beads 3 times with 1 mL of cold Tris-buffered saline (TBS) (50 mM Tris-HCl pH 7.5, 150 mM NaCl).</li>
	<li>Add 200 &#181;L of TBS to beads and appropriate amount of antibody. Rotate at 8 rpm overnight at 4 &#176;C. 
	NOTE: For many histone PTM antibodies, 1-3 &#181;g of antibody per IP is sufficient. However, titration experiments are recommended to determine appropriate concentrations. Recommended concentrations for well-characterized, commercial histone PTM antibodies are often accurate and can be found in published reports or product literature.</li>
	<li>Place the beads in a magnetic stand and remove the supernatant. Wash them with 1 mL of TBS.</li>
	<li>Resuspend the beads in 20 &#181;L of TBS per IP sample plus an additional 5 &#181;L (in case of pipetting error) of TBS. 
	NOTE: The beads can be stored short-term at 4 &#176;C until use.</li>
</ol>
2. Grow Yeast Cells
<ol>
	<li>Inoculate 10 mL of YPD (10% yeast extract, 20% peptone and 20% dextrose) with a single colony of the appropriate strain and grow it at 30 &#176;C overnight in a shaking incubator at 220 rpm.</li>
	<li>Measure the optical density at 600 nm (OD600) of the yeast culture using a spectrophotometer. Dilute the culture to an OD600 = 0.2 in 100 mL YPD in a new flask.</li>
	<li>Grow the cells at 30 &#176;C in a shaking incubator at 220 rpm until they reach mid-log phase (OD600 = 0.6-0.8).</li>
</ol>
3. In Vivo Crosslinking of Proteins to DNA
<ol>
	<li>When cultures reach mid-log phase, add 2.7 mL of 37% formaldehyde directly to the medium, for a final concentration of 1% formaldehyde. Transfer the flask to a shaker at 25 &#176;C (or room temperature) and shake it at 50 rpm for 15 min.</li>
	<li>Add 5 mL of 2.5 M glycine to the medium and continue shaking at 50 rpm at room temperature for 5 min to quench the formaldehyde.</li>
	<li>Transfer the cells to centrifuge bottles and spin the cells at 5800 x g for 5 min at 4 &#176;C. Decant supernatant.
	<ol>
		<li>Wash the cells by resuspending them in 45 mL of cold TBS and transfer the suspension into a 50 mL conical tube. Spin the tube at 2800 x g for 3 min at 4 &#176;C. Remove the supernatant.</li>
		<li>Wash the cells in 10 mL of cold chIP Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% nonidet octyl phenoxypolyethoxylethanol (NP-40), stored at 4 &#176;C).</li>
		<li>Spin the cells at 2800 x g for 3 min at 4 &#176;C. Remove the supernatant. 
		NOTE: Cells can be frozen with liquid nitrogen and stored at -80 &#176;C until further processing.</li>
	</ol>
	</li>
</ol>
4. Make Yeast Lysates
<ol>
	<li>Resuspend the cells in 1 mL of cold ChIP Lysis Buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1:1,000 dilution of the yeast protease inhibitor cocktail. Transfer the suspension to a 2.0 mL screw cap tube containing 200 &#181;L of glass beads.</li>
	<li>Transfer the cells to a bead beating apparatus for high speed agitation at 4 &#176;C and bead beat 6 times for 30 s each. Keep the cells on ice for at least 1 min between each bead beating.</li>
	<li>Transfer the lysate to a 1.5 mL tube using gel loading tips. Centrifuge the lysate at 15,500 x g for 20 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet in 250 &#181;L of MNase Digestion Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 21 mM CaCl2, 1% NP-40, stored at 4 &#176;C) by gently pipetting up and down.</li>
	<li>Add 2.5 &#181;L of MNase to the reactions. Mix them gently by inverting 4-6 times and immediately incubate it in a 37 &#176;C water bath for 20 min. 
	NOTE: Digest conditions should be optimized when a new stock of MNase is used. See step 10 for the protocol.</li>
	<li>Immediately place the tubes on ice to stop the reaction and add 5 &#181;L of 0.5 M EDTA for a final concentration of 10 mM EDTA. Mix them gently by inversion.</li>
	<li>Centrifuge the reaction at 15,500 x g for 15 min at 4 &#176;C and transfer the supernatant to a new 1.5 mL tube. 
	NOTE: Soluble (digested) chromatin will be in the supernatant. The pellet contains anything undigested and insoluble cell debris.</li>
</ol>
5. Immunoprecipitate (IP) Modified Histones
<ol>
	<li>Determine the protein concentration of each MNase-released chromatin fraction using a Bradford assay. Add 1 mL of Bradford reagent to each of 8 disposable cuvettes plus 1 additional cuvette for each protein sample to be tested.
	<ol>
		<li>Prepare a stock solution of 2 mg/mL bovine serum albumin (BSA).</li>
		<li>Add the appropriate volume to achieve the following concentrations of BSA in 1 mL of Bradford reagent: 0 &#956;g/mL, 0.5 &#956;g/mL, 1 &#956;g/mL, 2 &#956;g/mL, 4 &#956;g/mL, 6 &#956;g/mL, 8 &#956;g/mL and 10 &#956;g/mL. Add 2 &#956;L of the MNase-released chromatin fraction to the additional cuvettes.</li>
		<li>Cover the top of each cuvette with a small piece of parafilm and invert the cuvettes 4-6 times to mix the solution well. Incubate the cuvettes at room temperature for 15 min.</li>
		<li>Using a visible light spectrophotometer, measure the absorbance at 595 nm for the standard curve and experimental samples. 
		Note: Use the cuvette without BSA (0 &#956;g/mL) to blank the spectrophotometer prior to taking the first measurement.</li>
		<li>Plot a standard curve using the absorbance values for the different concentrations of BSA on software associated with the spectrophotometer or spreadsheet software. Based on the standard curve and the dilution factor used (1:500), use the absorbance values to calculate the protein concentration for each of the chromatin fraction samples.</li>
	</ol>
	</li>
	<li>For each IP, add 50 &#181;g of total protein from the MNase-released chromatin fraction to ChIP Lysis Buffer to reach a final volume of 1 mL. 
	NOTE: If setting up more than one IP per lysate, make a master mix of the lysate and ChIP Lysis Buffer and aliquot 1 mL to individual tubes for the IP.</li>
	<li>Remove 100 &#181;L from the IP mix to process as the input sample. Freeze the input sample at -20 &#176;C until step 7.1. 
	NOTE: Any remaining chromatin sample can also be frozen at -20 &#176;C and used for a subsequent IP if desired.</li>
	<li>Add 20 &#181;L of magnetic Protein A/G beads pre-bound with antibody to each IP sample. Rotate the sample at 8 rpm for 3 h (standard) or overnight (if preferred) at 4 &#176;C.</li>
</ol>
6. Wash IPs and Elute Histone-DNA Complexes
<ol>
	<li>Place the tubes in a magnetic stand and let beads collect on the side of the tube. Remove the supernatant using a pipette. Wash the beads successively with 1 mL of each of the buffers below.
	<ol>
		<li>Perform each wash by rotating at 8 rpm for 5 min at 4 &#176;C, placing beads in a magnetic stand, removing supernatant, and adding next buffer: 2x - ChIP Lysis Buffer, 2x - High Salt Wash Buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% NP-40, stored at 4 &#176;C), 1x - LiCl/detergent Wash Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, stored at 4 &#176;C), 1x - TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA, stored at 4 &#176;C), and 1x - TE .</li>
		<li>With last TE wash, transfer beads to a new tube using 0.5 mL of TE to transfer most of the beads, then another 0.5 mL of TE to wash the tube and transfer remaining beads.</li>
	</ol>
	</li>
	<li>Add 250 &#181;L of freshly made ChIP Elution Buffer (1% SDS, 1 mM NaHCO3). Vortex the solution briefly. Rotate the samples at 8 rpm for 15 min at room temperature.</li>
	<li>Place tube in a magnetic stand and collect the beads. Carefully transfer the supernatant to a new tube and avoid the beads. 
	NOTE: The eluate contains immunoprecipitated proteins and associated DNA.</li>
	<li>Add another 250 &#181;L of ChIP Elution Buffer to beads. Rotate the samples at 8 rpm for 15 min at room temperature.</li>
	<li>Carefully transfer the supernatant to the tube containing the first elution. If necessary, remove a smaller volume (240 &#181;L) for all IPs to avoid disturbing the beads.</li>
</ol>
7. Reverse Protein-DNA Crosslinks
<ol>
	<li>Thaw the input samples and add 400 &#181;L of ChIP Elution Buffer to each input.</li>
	<li>To both input and IP samples, add 20 &#181;L of 5 M NaCl, 5 &#181;L of 20 mg/mL glycogen, and 12.5 &#181;L of 20 mg/mL Proteinase K. Mix well by inverting or flicking tubes. Incubate the samples in a 65 &#176;C water bath overnight.</li>
</ol>
8. Purify and Concentrate DNA
<ol>
	<li>Add 10 &#181;L of 10mg/mL RNase A to the input and IP samples. Incubate the samples in a 37 &#176;C water bath for 30 min.</li>
	<li>Add 600 &#181;L of phenol:chlorofrom:isoamyl alcohol (25:24:1 PCI) to the aqueous solution and mix them well. Spin them at 15,500 x g for 5 min at room temperature.
	<ol>
		<li>Remove the aqueous layer to a new tube. Add 600 &#181;L of PCI and mix them well. Spin the tube at 15,500 x g for 5 min at room temperature. Transfer the aqueous layer to a new tube. 
		CAUTION: Work in the fume hood and wear appropriate personal protective equipment when working with PCI. Dispose liquid and solid waste as instructed by institutional guidelines.</li>
	</ol>
	</li>
	<li>Add 0.1x volume of 3M sodium acetate and 1 mL of cold 100% ethanol to precipitate the DNA. Invert the tube 4-6 times to mix the solution well. Incubate at -20 &#176;C overnight.
	<ol>
		<li>Spin the tube at 15,500 x g for 20 min at 4 &#176;C. Carefully remove the supernatant with a pipette.</li>
		<li>Wash the pellet in 1 mL of cold 70% ethanol. Spin it at 15,500 x g for 10 min at 4 &#176;C. Carefully remove the supernatant with a pipette.</li>
		<li>Air-dry the pellets in the hood for approximately 20 min (until completely dry). Resuspend IP samples in 50 &#181;L of nuclease-free water and resuspend input samples in 100 &#181;L of nuclease-free water. Store the DNA samples at -20 &#176;C until performing qPCR.</li>
	</ol>
	</li>
</ol>
9. Quantitative PCR (qPCR) to Detect Enriched Genomic Regions
<ol>
	<li>Make a qPCR master mix for each set of primers. One reaction contains 5 &#181;L of 2x qPCR mix, 0.25 &#181;L of 20 &#181;M forward primer, and 0.25 &#181;L of 20 &#181;M reverse primer. 
	NOTE: Proper primer design and testing for qPCR experiments are described elsewhere14,15,16.</li>
	<li>Make DNA master mixes for each DNA sample. One reaction contains 0.5 &#181;L of DNA and 4.0 &#181;L of nuclease-free water.</li>
	<li>Add 5.5 &#181;L of the appropriate primer master mix and 4.5 &#181;L of the appropriate DNA master mix to each well on a 384-well qPCR plate. 
	Note: Start with adding 5.5 &#181;L of the primer mix to each well. Spin the plate at 200 x g for 1 min at room temperature before adding 4.5 &#181;L of the DNA mix.</li>
	<li>Adhere the seal to the plate and spin at 200 x g for 1 min at room temperature.</li>
	<li>Perform qPCR using a real-time qPCR system with the following conditions: 95 &#176;C for 3 min; 40 cycles of 95 &#176;C for 10 s, 55 &#176;C for 30 s; melting curve 65 &#176;C to 95 &#176;C with 0.5 &#176;C increments, 5 s.</li>
	<li>For each primer pair, calculate the percent input of the IP sample relative to the 5% input sample used in the qPCR. Use the means of the technical PCR triplicates to perform the calculations. 
	NOTE: If substantial variation is observed in the PCR triplicates, it is best to repeat the qPCR with attention to master mix composition, pipetting errors and cross-contamination between wells in the 384-well plate.
	<ol>
		<li>Adjust the Cq values for the input to 100% by subtracting the number of cycles representing the dilution factor from the raw Cq values. 
		NOTE: Five percent input represents a dilution factor of 20-fold, which equals 4.32 cycles (log2 20 = 4.32).</li>
		<li>Calculate the percent input as 2^(Cqinput - CqIP) multiplied by 100.</li>
	</ol>
	</li>
</ol>
10. Determine MNase Digest Conditions (Recommended Prior to First Full chIP Experiment)
<ol>
	<li>Follow steps 2.1 through 4.4 of the preceding protocol. Scale up the protocol to generate enough lysate for multiple samples of MNase digestion of the chromatin-containing pellet. At step 4.4, resuspend the pellets in 1.25 mL of MNase Digestion Buffer. Aliquot the samples to 5 tubes so that each contains 250 &#181;L of the chromatin pellet resuspended in MNase Digestion Buffer.</li>
	<li>Add 0, 1.5, 2.5 or 5 &#181;L of MNase to each tube. Mix them gently by inverting 4-6 times and immediately incubate the tubes in a 37 &#176;C water bath for 20 min.</li>
	<li>Stop reactions by immediately placing tubes on ice and adding 5 &#181;L of 0.5 M EDTA for a 10 mM final concentration. Mix the solution gently by inversion.</li>
	<li>Centrifuge the tubes at 15,500 x g for 15 min at 4 &#176;C and transfer the supernatant to a new 1.5 mL tube.</li>
	<li>Add SDS to 1% final concentration (25 &#181;L of 10% stock solution) and NaHCO3 to 0.1 M final concentration (25 &#181;L of 1M stock solution) to the samples in the 1.5 mL tubes.</li>
	<li>Add 20 &#181;L of 5M NaCl, 5 &#181;L of glycogen, and 12.5 &#181;L of 20mg/mL proteinase K to the samples in the 1.5mL tubes. Incubate them at 65 &#176;C overnight.</li>
	<li>Add 10 &#181;L of 10 mg/mL RNaseA. Incubate at 37 &#176;C for 30 min.</li>
	<li>Add 300 &#181;L of PCI to the aqueous solution and mix them well. Spin at 15,500 x g for 5 min at room temperature.
	<ol>
		<li>Remove aqueous layer to a new tube. Add 300 &#181;L of PCI and mix well. Spin at 15,500 x g for 5 min at room temperature. Transfer aqueous layer to a new tube.</li>
	</ol>
	</li>
	<li>Add 0.1x volume of 3 M sodium acetate (usually ~30 &#181;L) and 1 mL of cold 100% ethanol to precipitate the DNA. Invert the tube 4-6 times to mix well. Incubate the tube at -80 &#176;C for 1 h or -20 &#176;C overnight.
	<ol>
		<li>Spin the tube at 15,500 x g for 20 min at 4&#176;C. Carefully remove supernatant with a pipette.</li>
		<li>Wash the pellets in 1 mL of cold 70% ethanol. Spin at 15,500 x g for 10 min at 4 &#176;C.</li>
		<li>Let the pellets air-dry in the hood approximately 20 min (or until completely dry). Resuspend the pellets in 30 &#181;L of nuclease-free water.</li>
	</ol>
	</li>
	<li>Add the DNA loading dye and run 20 &#181;L on a 2% agarose gel to visualize digested DNA.</li>
</ol>

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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></ol>

The authors have nothing to disclose.

The procedure described here allows for the efficient recovery of DNA associated with modified histones in yeast cells by immunoprecipitation. This is followed by qPCR using primers which amplify regions of interest to determine local enrichment or depletion of specific histone modifications. Despite being developed as a method almost 20 years ago, chIP remains the defining assay for investigating histone modification status at different genomic regions and under diverse conditions. Although chIP coupled to next generati...

The dynamic post-translational modification (PTM) of histones is a key regulatory mechanism for many DNA-templated processes, including transcription, replication and DNA repair1,2. The ability to determine the abundance and precise localization of modified histones concomitant with these processes is therefore critical to understanding their regulation under different conditions in the cell. The development of chromatin immunoprecipitation (chIP) as a method largely stemmed from biochemical studies of the interactions of proteins with DNA, particularly in vitro methods using chemical crosslinkers, coupled with the need to evaluate the dynamic nature of protein-DNA interactions in vivo and at specific regions of the genome3,4,5. The advancement of quantitative PCR (qPCR) and sequencing technologies has also expanded the ability to perform chIP experiments with quantitative comparisons and across whole genomes, making it a powerful tool for dissecting DNA-protein interactions at multiple levels.
Currently, chIP is a required method for any research group interested in chromatin-mediated regulation of the genome as there are no comparable methods for directly interrogating the physical link between a modified histone and a specific genomic locus in vivo. Although variations of this method using next generation sequencing to map histone modifications throughout the genome6,7 are available, these approaches may address different scientific questions and their scale, cost and technical resources may be limiting for some research groups. Additionally, targeted chIP-qPCR is necessary to complement these approaches by providing methods to both optimize the chIP protocol prior to sequencing and to validate results from the epigenomic datasets. Mass spectrometry based approaches for identifying the full complement of histone marks associated with genomic regions have also emerged8,9,10,11, however, these approaches have some limitations regarding which regions of the genome can be probed and they require technical expertise and instrumentation that will not be available to all research groups. Therefore, chIP remains a foundational method for analyzing the abundance and distribution of histone modifications under diverse conditions for all research groups interested in epigenetics, chromatin and the regulation of genomic functions.
Here, we describe a method for chIP using the budding yeast model Saccharomyces cerevisiae (S. cerevisiae) to investigate the distribution of histone PTMs at chromatin. This approach relies on a number of core components of chIP protocols developed in yeast and also applied to diverse model systems12,13. Interactions between modified histones and DNA in the cell are preserved by crosslinking with formaldehyde. Following lysate preparation, chromatin fragments are solubilized into uniformly-sized fragments by digestion with micrococcal nuclease. Immunoprecipitation of the modified histones is performed with either commercial or lab-generated antibodies and any associated DNA is isolated and analyzed for enrichment at particular genomic regions using qPCR (Figure 1). For many histone modifications, the quantity of DNA obtained from this protocol is sufficient for testing more than 25 different genomic loci by qPCR.
This chIP method is highly versatile for monitoring the distribution of a single histone modification across multiple mutant strains or environmental conditions, or for testing multiple histone modifications in wildtype cells at a number of genomic loci. Furthermore, numerous components of the protocol are easily adjustable to optimize detection of either highly- or lowly-abundant histone marks. Finally, performing chIP of modified histones in budding yeast provides the opportunity to use key controls for antibody specificity that are largely unavailable in other systems. Namely, yeast strains can be generated that carry point mutations in histone residues that are targeted for modification, and, in some cases, there is only a single enzyme that catalyzes modification on a particular histone residue (e.g. histone lysine methyltransferases). Therefore, chIP can be performed in either the histone mutant or enzyme deletion strains to assay the extent to which non-specific binding of the antibody may be occurring and generating false positive results. This control is particularly valuable for newly-developed antibodies, and may even be used to validate antibody specificity for conserved histone modifications prior to their use in other systems. This approach complements other methods to test antibody specificity that distinguish among different modification states (such as mono-, di- and tri-methylation), including probing arrays of modified peptides and performing western blots of histones or nucleosomes with defined modifications. Overall, chIP in budding yeast is a powerful method for assessing the dynamics of histone PTMs throughout the genome and dissecting the mechanisms governing their regulation.

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			<td>P50220-0.1</td>
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			<td>R6513-50MG</td>
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			<td style="width:164px;">23238</td>
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			<td>23210</td>
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			<td>ABE532</td>
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			<td height="20" style="height:20px;width:244px;">Protein A/G Magnetic Beads</td>
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			<td>88803</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">magnetic stand for 1.5mL tubes</td>
			<td>Fisher Scientific</td>
			<td>PI-21359</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Acid-Washed Glass Beads</td>
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			<td style="width:164px;">G8772</td>
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			<td height="40" style="height:40px;width:244px;">2.0 mL screw-cap tubes with sealing rings</td>
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			<td>Z763837-1000EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Gel loading tips</td>
			<td>Fisher Scientific</td>
			<td>07-200-288</td>
			<td></td>
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			<td height="20" style="height:20px;width:244px;">Cuvettes</td>
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			<td style="width:164px;">50-476-476</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Parafilm</td>
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			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">50 mL conical tubes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">384-Well PCR Plate</td>
			<td>Fisher Scientific</td>
			<td>AB-1384W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Gyratory Floor Shaker</td>
			<td style="width:301px;">New Brunswick Scientific</td>
			<td style="width:164px;">Model G10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Spectrophotometer</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">ND-2000c</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">&#160;Real-Time PCR Detection System</td>
			<td style="width:301px;">Bio-Rad</td>
			<td style="width:164px;">1855485</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

One key component of this protocol is optimizing the concentration of micrococcal nuclease (MNase) used to digest the chromatin into soluble fragments, as outlined in Step 10. This is critical for obtaining high resolution data regarding the distribution of histone modifications at genomic regions of interest. An MNase titration should be performed to determine the most suitable concentration to achieve primarily mono-nucleosomes with a smaller amount of di-nucleosomes in the soluble chro...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

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.

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 ...

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12.6K Views.  University of Maryland. 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. 

Video: Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

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. ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

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.

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.

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

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, ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

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

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

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

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

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

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

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

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription factors, histone modifications, and other DNA-associated proteins. ChIP-seq data can also be used to find differential binding of transcription factors in different environmental conditions or cell types. Initially, ChIP was performed through hybridization on a microarray (ChIP-chip); however, ChIP-seq has become the preferred method through technological advancements, decreasing financial barriers to sequencing, and massive amounts of high-quality data output. Techniques of performing ChIP-seq with bacterial biofilms, a major source of persistent and chronic infections, are described in this protocol. ChIP-seq is performed on Salmonella enterica serovar Typhimurium biofilm and planktonic cells, targeting the master biofilm regulator, CsgD, to determine differential binding in the two cell types. Here, we demonstrate the appropriate amount of biofilm to harvest, normalizing to a planktonic control sample, homogenizing biofilm for cross-linker access, and performing routine ChIP-seq steps to obtain high quality sequencing results.

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a method used to establish interactions between transcription factors and the genomic sequences they control. This protocol outlines techniques for performing ChIP-seq with bacterial biofilms, using Salmonella enterica serovar Typhimurium bacterial biofilm as an example.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

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 ...