A.L. was supported by a Canadian Graduate Scholarship from the Canadian Institutes of Health Research. This work was supported by grants from Genome British Columbia and the Canadian Institutes of Health Research (CIHR-120589) &#160;as part of the Canadian Epigenetics, Environment and Health Research Consortium Initiative and by the Terry Fox Research Institute Program Project Grant #TFF-122869 to M.H. and a Terry Fox Research Institute New Investigator Award (Grant # 1039) to M.H.

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+guidelines+and+practices+of+the+ENCODE+and+modENCODE+consortia.">ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.</a> Genome Res. 22 (9), 1813-1831 (2012).</li><li>Heinz, 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=Simple+Combinations+of+Lineage-Determining+Transcription+Factors+Prime+cis-Regulatory+Elements+Required+for+Macrophage+and+B+Cell+Identities.">Simple Combinations of Lineage-Determining Transcription Factors Prime cis-Regulatory Elements Required for Macrophage and B Cell Identities.</a> Mol. Cell. 38 (4), 576-589 (2010).</li><li>Feng, J., Liu, T., Qin, B., Zhang, Y., Liu, X. 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The authors declare that they have no competing financial interests.

Given the combinatorial nature of chromatin modification and nucleosome positioning in transcriptional regulation, a method that enables simultaneous measurements of these features is likely to provide new insights into epigenetic regulation. The ndChIP-seq protocol presented here is a native ChIP-seq protocol optimized to enable simultaneous interrogation of histone modification and nucleosome density in rare primary cells9,10. ndChIP-seq utilizes enzymatic digestion of chromatin that, when coupled to paired-end massively parallel sequencing and a Gaussian mixture distribution model, allows for the investigation histone modifications at single nucleosome level and the deconvolution of epigenomic profiles driven by heterogeneity within a population. Using this protocol, we have previously reported a unique distribution of immuno-precipitated fragment sizes, determined by paired-end read boundaries, associated with specific chromatin states defined by ChromHMM10,24.
The quality of a ndChIP-seq library depends on multiple factors, such as antibody specificity and sensitivity, optimal MNase digestion conditions, and quality of the chromatin. The specificity of the antibodies used is crucial in producing successful ndChIP-seq library. An ideal antibody shows high affinity against the epitope of interest with little cross reactivity with other epitopes. It is equally important to choose magnetic beads with the highest affinity for the antibody of choice.
MNase digestion is a critical and time- and concentration-sensitive reaction in this protocol. Therefore, when processing multiple samples, it is important that each reaction is incubated for an equivalent amount of time (see step 2.2). The quality of the chromatin is another factor that significantly effects the outcome of ndChIP-seq. Fragmented chromatin leads to a sub-optimal MNase digestion profile and results in libraries with a low signal to noise ratio. Primary samples with low cell viability or degraded chromatin, such as formalin-fixed paraffin-embedded (FFPE) tissue are not recommended for this protocol.
The addition of a PIC during chromatin extraction reduces undesired (i.e., random) chromatin fragmentation and preserves integrity of histone tails. As such, PIC needs to be added to lysis buffer and immunoprecipitation buffer just prior to use. While selecting cells via flow cytometry, select for viable cells and ensure cells are sorted at a low flow rate to increase the accuracy of the cell number estimate and viability of cells. Avoid sorting cells directly into lysis buffer. The sheath buffer will dilute the lysis buffer and prevents effective permeabilization of the cell membrane to MNase. Depending on a type of cells or organism, titration of MNase may be required to obtain optimal digestion.
ndChIP-seq on mammalian cells requires a minimum sequencing depth of 50 million paired-reads (25 million fragments) for narrow marks and 100 million paired-reads (50 million fragments) for broad marks and input. The Gaussian mixture distribution algorithm will not perform optimally on libraries that have been sequenced to a depth significantly below this recommendation. ndChIP-seq will not classify promoters with little separation between the weighted distribution value for mono- and di-nucleosome fragment lengths into mono- or di-nucleosome dominated promoters. Therefore, these promoters must be removed in the subsequent analysis. Biological replicates can be generated to increase confidence in predicted distributions and identify technical variability in the MNase digestion and library construction.
Unlike previous iterations of native ChIP-seq protocols, ndChIP-seq provides the means to investigate combinatorial effect of chromatin structure and histone modification by utilizing fragment size post immunoprecipitation to integrate nucleosome density, determined by MNase accessibility, with histone modification measurements. Application of ndChIP-seq to primary cells and tissues will provide novel insights into the integrative nature of epigenetic regulation and permit identification of epigenetic signatures due to heterogeneity within the population.

The eukaryotic genome is packaged into chromatin via repeating nucleosome structures that consist of two copies of four histone proteins (e.g., H2A, H2B, H3, and H4) circumscribed by 146 base pairs of DNA1,2. Chromatin remodeling complexes control nucleosome position within gene promoter boundaries and participate in the regulation of gene expression by altering accessibility of the DNA to transcription factors and to the RNA polymerase machinery3,4.
Amino terminal tails of histones within the nucleosome are subjected to various covalent modifications, including acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, formylation, and hydroxylation of specific amino acids5,6,7,8. Positions and degrees of these modifications dictate a chromatin state that influence chromatin structure and control access of the molecular complexes that allow activation of transcription7. Given that both nucleosome density and histone modification play a role in the local control of gene transcription, we developed a native ChIP approach that enables the simultaneous measurement of nucleosome density and histone modification9,10.
Native ChIP-seq takes advantage of the endonuclease micrococci nuclease (MNase) to digest intact chromatin in its native state within the nucleus11,12, a property that has been leveraged to map nucleosome positioning13,14,15. Nucleosome density ChIP-seq (ndChIP-seq) takes advantage of the property of preferential access of MNase to open regions of chromatin to generate measurements that combine MNase accessibility with histone modification10. ndChIP-seq is suitable for the profiling of histone modifications in rare primary cells, tissues, and cultured cells. Here, we present a detailed protocol that enables the generation of sequence datasets suitable for a previously described analytical frame work10 that integrates fragment size post immunoprecipitation, determined by paired-end read boundaries, to simultaneously investigate MNase accessibility with histone modification measurements. Previously, application of this protocol to 10,000 primary human cord blood derived CD34+ cells and human embryonic stem cells revealed unique relationships between chromatin structure and histone modifications within these cell types10. Given its ability to simultaneously measure nucleosome accessibility and histone modification, ndChIP-seq is capable of revealing epigenomic features in a cell population at a single nucleosome level, and resolving heterogeneous signatures into their constituent elements. An example of the exploration of heterogeneous cellular populations by ndChIP-seq is investigation of bivalent promoters, where both H3K4me3, an active mark, and H3K27me3, a repressive mark, are present10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	
	<tbody>
		<tr >
			<td >1M Tris-HCl &#8211;pH 7.5</td>
			<td >Thermo Scientific</td>
			<td >15567-027</td>
			<td ></td>
		</tr>
		<tr >
			<td >1M Tris-HCl &#8211;pH 8</td>
			<td >Thermo Scientific</td>
			<td >15568-025</td>
			<td></td>
		</tr>
		<tr >
			<td >0.5M EDTA</td>
			<td >Thermo Scientific</td>
			<td >AM9260G</td>
			<td></td>
		</tr>
		<tr >
			<td >5M NaCl</td>
			<td >Sigma</td>
			<td >1001385276</td>
			<td></td>
		</tr>
		<tr >
			<td >Triton X-100</td>
			<td >Sigma</td>
			<td >1001412354</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium-Deoxycholate</td>
			<td >Sigma</td>
			<td >1001437582</td>
			<td></td>
		</tr>
		<tr >
			<td >SDS</td>
			<td >Thermo Scientific</td>
			<td >15525-017</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium-Bicarbonate</td>
			<td >Fisher Scientific</td>
			<td >S233-500</td>
			<td></td>
		</tr>
		<tr >
			<td >100% EtOH</td>
			<td >NA</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >V-bottom 96 well plate (AB 1400)</td>
			<td >Thermo Scientific</td>
			<td >AB-1400-L</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P2 pipetman</td>
			<td >Mandel</td>
			<td >F144801</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P10 pipetman</td>
			<td >Mandel</td>
			<td >F144802</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P20 pipetman</td>
			<td >Mandel</td>
			<td >F123600</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P100 pipetman</td>
			<td >Mandel</td>
			<td >F123615</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P200 pipetman</td>
			<td >Mandel</td>
			<td >F123601</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P1000 pipetman</td>
			<td >Mandel</td>
			<td >F123603</td>
			<td></td>
		</tr>
		<tr >
			<td >Micrococcal Nuclease&#160;</td>
			<td >NEB</td>
			<td >M0247S</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo Mixer C (Heating block mixer)</td>
			<td >Eppendorf</td>
			<td >5382000023</td>
			<td></td>
		</tr>
		<tr >
			<td >Multi 12-channel Pippet P20</td>
			<td >Rainin</td>
			<td >17013803</td>
			<td></td>
		</tr>
		<tr >
			<td >Multi 12-channel Pippet P200</td>
			<td >Rainin</td>
			<td >17013805</td>
			<td></td>
		</tr>
		<tr >
			<td >1.5 ml LoBind tube</td>
			<td >Eppendorf</td>
			<td >22431021</td>
			<td></td>
		</tr>
		<tr >
			<td >0.5 ml LoBind tube</td>
			<td >Eppendorf</td>
			<td >22431005</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic Plate Cover</td>
			<td >Edge Bio</td>
			<td >48461</td>
			<td></td>
		</tr>
		<tr >
			<td >PCR cover</td>
			<td >Bio Rad</td>
			<td >MSD-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Aluminum Plate Cover</td>
			<td >Bio Rad</td>
			<td >MSF-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Elution buffer (EB buffer)</td>
			<td >Qiagen</td>
			<td >19086</td>
			<td></td>
		</tr>
		<tr >
			<td >1M DTT</td>
			<td >Sigma</td>
			<td >646563</td>
			<td></td>
		</tr>
		<tr >
			<td >Eppendorf 5810R centrifuge&#160;</td>
			<td >Eppendorf</td>
			<td >22625004</td>
			<td></td>
		</tr>
		<tr >
			<td >Ultrapure water</td>
			<td >Thermo Scientific</td>
			<td >10977-015</td>
			<td></td>
		</tr>
		<tr >
			<td >Protein G dynabeads</td>
			<td >Thermo Scientific</td>
			<td >10001D</td>
			<td></td>
		</tr>
		<tr >
			<td >Protein A dynabeads</td>
			<td >Thermo Scientific</td>
			<td >10001D</td>
			<td></td>
		</tr>
		<tr >
			<td >Protease inhibitor Cocktail</td>
			<td >Calbiochem</td>
			<td >539134</td>
			<td></td>
		</tr>
		<tr >
			<td >Rotating platform</td>
			<td >Mandel</td>
			<td >1b109-12052010</td>
			<td></td>
		</tr>
		<tr >
			<td >Plate magnet</td>
			<td >Alpaqua</td>
			<td >2523</td>
			<td></td>
		</tr>
		<tr >
			<td >Domed 12-cap strip</td>
			<td >Bio Rad</td>
			<td >TCS1201</td>
			<td></td>
		</tr>
		<tr >
			<td >Buffer G2</td>
			<td >Qiagen</td>
			<td >1014636</td>
			<td></td>
		</tr>
		<tr >
			<td >Protease</td>
			<td >Qiagen</td>
			<td >19155</td>
			<td></td>
		</tr>
		<tr >
			<td>Tube Magnet (DynaMag-2)</td>
			<td >Thermo Scientific</td>
			<td >12321D</td>
			<td></td>
		</tr>
		<tr >
		<td>Tube Magnet (DynaMag-2)</td>
			<td >LabCore</td>
			<td >730-006</td>
			<td></td>
		</tr>
		<tr >
			<td >V shape Plastic reservoir</td>
			<td >Mandel</td>
			<td >S0100A</td>
			<td></td>
		</tr>
		<tr >
			<td >Vortex</td>
			<td >Mandel</td>
			<td >C1801</td>
			<td></td>
		</tr>
		<tr >
			<td >Mini Fuge</td>
			<td >Mettler Toledo</td>
			<td >XS2035</td>
			<td></td>
		</tr>
		<tr >
			<td >Analytical scale</td>
			<td >Mandel</td>
			<td >LB109-12052010</td>
			<td></td>
		</tr>
		<tr >
			<td >Quick Ligation Reaction Buffer, 5X</td>
			<td >NEB</td>
			<td>B6058S</td>
			<td></td>
		</tr>
		<tr >
			<td >DNA Polymerase I, Large (Klenow) Fragment</td>
			<td >NEB</td>
			<td>M0210S</td>
			<td></td>
		</tr>
		<tr >
			<td >Klenow Fragment (3&#39;-5&#39; exo)</td>
			<td >NEB</td>
			<td>M0212S</td>
			<td></td>
		</tr>
		<tr >
			<td >T4 DNA Polymerase</td>
			<td >NEB</td>
			<td>M0203S</td>
			<td></td>
		</tr>
		<tr >
			<td >T4 Polynucleotide Kinase</td>
			<td >NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr >
			<td >Deoxynucleotide Solution mix, 10mM</td>
			<td >NEB</td>
			<td>N0447S</td>
			<td></td>
		</tr>
		<tr >
			<td >dATP Solution 10mM</td>
			<td >NEB</td>
			<td>N0440S</td>
			<td></td>
		</tr>
		<tr >
			<td >Quick T4 DNA Ligase</td>
			<td >NEB</td>
			<td>M0202T</td>
			<td></td>
		</tr>
		<tr >
			<td >Adenosine 5&#39;-Triphosphate (ATP), 25mM</td>
			<td >NEB</td>
			<td>P0756S</td>
			<td></td>
		</tr>
		<tr >
			<td >DNA Polymerase</td>
			<td >Thermo Scientific</td>
			<td>F549L</td>
			<td></td>
		</tr>
		<tr >
			<td >NEBuffer 2</td>
			<td >NEB</td>
			<td>B7002S</td>
			<td></td>
		</tr>
		<tr >
			<td >Hank&#39;s buffered salt solution</td>
			<td >Thermo Scientific</td>
			<td>14025076</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal bovine serum&#160;</td>
			<td >Thermo Scientific</td>
			<td>A3160702</td>
			<td></td>
		</tr>
		<tr >
			<td >phosphate buffer saline&#160;</td>
			<td >Thermo Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K4me3</td>
			<td >Cell Signaling</td>
			<td >9751S</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K4me1</td>
			<td >Diagenode</td>
			<td >C15410037</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K27me3</td>
			<td >Diagenode</td>
			<td >pAb-069-050</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K36me3</td>
			<td >Abcam</td>
			<td >ab9050</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K9me3</td>
			<td >Diagenode</td>
			<td >C15410056</td>
			<td></td>
		</tr>
		<tr >
			<td >SeraMag&#160; super-paramagnetic beads</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis at JoVE.com

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

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 ...

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JoVE Journal

Genetics

22.0K Views.  University of British Columbia. 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.

Video: Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

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 ...

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

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. ...

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 ...

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, ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

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 ...

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 ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...