Name: generation of native chromatin immunoprecipitation sequencing libraries for nucleosome density analysis
Uploaded: 2017-12-12T12:30:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis at JoVE.com

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>
	<li>Simpson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+Positioning:+Occurrence,+Mechanisms,+and+Functional+Consequences.">Nucleosome Positioning: Occurrence, Mechanisms, and Functional Consequences.</a> Prog. Nucleic Acid Res. Mol. Biol. 40, 143-184 (1991).</li><li>Kornberg, R. D., Lorch, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-five+years+of+the+nucleosome,+fundmamental+particle+of+the+eukaryotic+chromosome.">Twenty-five years of the nucleosome, fundmamental particle of the eukaryotic chromosome.</a> Cell. 98, 285-294 (1999).</li><li>Schones, D. E., 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+regulation+of+nucleosome+positioning+in+the+human+genome.">Dynamic regulation of nucleosome positioning in the human genome.</a> Cell. 132 (5), 887-898 (2008).</li><li>Segal, E., 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+genomic+code+for+nucleosome+positioning.">A genomic code for nucleosome positioning.</a> Nature. 442 (7104), 772-778 (2006).</li><li>Shiio, Y., Eisenman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+sumoylation+is+associated+with+transcriptional+repression.">Histone sumoylation is associated with transcriptional repression.</a> PNAS. 100 (23), 13225-13230 (2003).</li><li>Li, L., Lorzadeh, A., Hirst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+variation:+An+emerging+vantage+point+for+cancer+biology.">Regulatory variation: An emerging vantage point for cancer biology.</a> Wiley Interdiscip. Rev. Syst. Biol. Med. 6 (1), 37-59 (2014).</li><li>Jiang, 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=Investigation+of+the+acetylation+mechanism+by+GCN5+histone+acetyltransferase.">Investigation of the acetylation mechanism by GCN5 histone acetyltransferase.</a> PLoS ONE. 7 (5), (2012).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methylation+in+transcriptional+control.">Histone methylation in transcriptional control.</a> Curr. Opin. Genet. Dev. 12 (2), 198-209 (2002).</li><li>Brind'Amour, J., Liu, S., Hudson, M., Chen, C., Karimi, M. M., Lorincz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultra-low-input+native+ChIP-seq+protocol+for+genome-wide+profiling+of+rare+cell+populations.">An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations.</a> Nat. Commun. 6, 6033 (2015).</li><li>Lorzadeh, 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=Nucleosome+Density+ChIP-Seq+Identifies+Distinct+Chromatin+Modification+Signatures+Associated+with+MNase+Accessibility.">Nucleosome Density ChIP-Seq Identifies Distinct Chromatin Modification Signatures Associated with MNase Accessibility.</a> Cell Rep. 17 (8), 2112-2124 (2016).</li><li>Noll, M., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+micrococcal+nuclease+on+chromatin+and+the+location+of+histone+H1.">Action of micrococcal nuclease on chromatin and the location of histone H1.</a> J. Mol. Biol. 109 (3), 393-404 (1977).</li><li>Skene, P. J., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+generating+high-resolution+maps+of+genome+wide+protein+binding.">A simple method for generating high-resolution maps of genome wide protein binding.</a> eLife. 4, 09225 (2015).</li><li>Brogaard, K., Xi, L., Wang, J. -. P., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+map+of+nucleosome+positions+in+yeast+at+base-pair+resolution.">A map of nucleosome positions in yeast at base-pair resolution.</a> Nature. , (2012).</li><li>Cui, K., Zhao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+approaches+to+determining+nucleosome+occupancy+in+metazoans+using+MNase-Seq.">Genome-wide approaches to determining nucleosome occupancy in metazoans using MNase-Seq.</a> Methods Mol. Biol. 833, 413-419 (2012).</li><li>Mieczkowski, 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=MNase+titration+reveals+differences+between+nucleosome+occupancy+and+chromatin+accessibility.">MNase titration reveals differences between nucleosome occupancy and chromatin accessibility.</a> Nat. Commun. 7, 11485 (2016).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Specific+Cell+Population+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS).</a> JOVE. (41), (2010).</li><li>Rohland, N., Reich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective,+high-throughput+DNA+sequencing+libraries+for+multiplexed+target+capture.">Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture.</a> Genome Res. 22 (5), 939-946 (2012).</li><li>Zheng, Y., Josefowicz, S. Z., Kas, A., Chu, T. T., Gavin, M. A., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+Foxp3+target+genes+in+developing+and+mature+regulatory+T+cells.">Genome-wide analysis of Foxp3 target genes in developing and mature regulatory T cells.</a> Nature. 445 (7130), 936-940 (2007).</li><li>Krishnakumar, R., Gamble, M. J., Frizzell, K. M., Berrocal, J. G., Kininis, M., Kraus, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+Binding+of+PARP-1+and+Histone+H1+at+Promoters+Specifies+Transcriptional+Outcomes.">Reciprocal Binding of PARP-1 and Histone H1 at Promoters Specifies Transcriptional Outcomes.</a> Science. 319 (5864), 819-821 (2008).</li><li>Mendenhall, E. M., 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=Locus-specific+editing+of+histone+modifications+at+endogenous+enhancers.">Locus-specific editing of histone modifications at endogenous enhancers.</a> Nat. Biotechnol. 31 (12), 1133-1136 (2013).</li><li>Landt, S. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+ChIP-seq+enrichment+using+MACS.">Identifying ChIP-seq enrichment using MACS.</a> Nat. Protoc. 7 (9), 1728-1740 (2012).</li><li>Fraley, C., Raftery, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCLUST:+Software+for+Model-Based+Cluster+Analysis.">MCLUST: Software for Model-Based Cluster Analysis.</a> J. Classif. 16 (2), 297-306 (1999).</li></ol>

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.

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			<td >1M Tris-HCl &#8211;pH 7.5</td>
			<td >Thermo Scientific</td>
			<td >15567-027</td>
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			<td >1M Tris-HCl &#8211;pH 8</td>
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			<td >0.5M EDTA</td>
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			<td >5M NaCl</td>
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			<td >Triton X-100</td>
			<td >Sigma</td>
			<td >1001412354</td>
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			<td >Sodium-Deoxycholate</td>
			<td >Sigma</td>
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			<td >Thermo Scientific</td>
			<td >15525-017</td>
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			<td >Sodium-Bicarbonate</td>
			<td >Fisher Scientific</td>
			<td >S233-500</td>
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			<td >100% EtOH</td>
			<td >NA</td>
			<td >NA</td>
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			<td >V-bottom 96 well plate (AB 1400)</td>
			<td >Thermo Scientific</td>
			<td >AB-1400-L</td>
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			<td >Gilson P2 pipetman</td>
			<td >Mandel</td>
			<td >F144801</td>
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			<td >Gilson P10 pipetman</td>
			<td >Mandel</td>
			<td >F144802</td>
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			<td >Gilson P20 pipetman</td>
			<td >Mandel</td>
			<td >F123600</td>
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			<td >Gilson P100 pipetman</td>
			<td >Mandel</td>
			<td >F123615</td>
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			<td >Gilson P200 pipetman</td>
			<td >Mandel</td>
			<td >F123601</td>
			<td></td>
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			<td >Gilson P1000 pipetman</td>
			<td >Mandel</td>
			<td >F123603</td>
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			<td >Micrococcal Nuclease&#160;</td>
			<td >NEB</td>
			<td >M0247S</td>
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			<td >Thermo Mixer C (Heating block mixer)</td>
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			<td >Multi 12-channel Pippet P20</td>
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			<td >Multi 12-channel Pippet P200</td>
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			<td >0.5 ml LoBind tube</td>
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			<td >Edge Bio</td>
			<td >48461</td>
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			<td >PCR cover</td>
			<td >Bio Rad</td>
			<td >MSD-1001</td>
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			<td >Aluminum Plate Cover</td>
			<td >Bio Rad</td>
			<td >MSF-1001</td>
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			<td >Elution buffer (EB buffer)</td>
			<td >Qiagen</td>
			<td >19086</td>
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			<td >1M DTT</td>
			<td >Sigma</td>
			<td >646563</td>
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			<td >Eppendorf 5810R centrifuge&#160;</td>
			<td >Eppendorf</td>
			<td >22625004</td>
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			<td >Ultrapure water</td>
			<td >Thermo Scientific</td>
			<td >10977-015</td>
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			<td >Protein G dynabeads</td>
			<td >Thermo Scientific</td>
			<td >10001D</td>
			<td></td>
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			<td >Protein A dynabeads</td>
			<td >Thermo Scientific</td>
			<td >10001D</td>
			<td></td>
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		<tr >
			<td >Protease inhibitor Cocktail</td>
			<td >Calbiochem</td>
			<td >539134</td>
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			<td >Rotating platform</td>
			<td >Mandel</td>
			<td >1b109-12052010</td>
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			<td >Plate magnet</td>
			<td >Alpaqua</td>
			<td >2523</td>
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		<tr >
			<td >Domed 12-cap strip</td>
			<td >Bio Rad</td>
			<td >TCS1201</td>
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			<td >Buffer G2</td>
			<td >Qiagen</td>
			<td >1014636</td>
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			<td >Protease</td>
			<td >Qiagen</td>
			<td >19155</td>
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		<tr >
			<td>Tube Magnet (DynaMag-2)</td>
			<td >Thermo Scientific</td>
			<td >12321D</td>
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		<tr >
		<td>Tube Magnet (DynaMag-2)</td>
			<td >LabCore</td>
			<td >730-006</td>
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			<td >V shape Plastic reservoir</td>
			<td >Mandel</td>
			<td >S0100A</td>
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		<tr >
			<td >Vortex</td>
			<td >Mandel</td>
			<td >C1801</td>
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		<tr >
			<td >Mini Fuge</td>
			<td >Mettler Toledo</td>
			<td >XS2035</td>
			<td></td>
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		<tr >
			<td >Analytical scale</td>
			<td >Mandel</td>
			<td >LB109-12052010</td>
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		<tr >
			<td >Quick Ligation Reaction Buffer, 5X</td>
			<td >NEB</td>
			<td>B6058S</td>
			<td></td>
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		<tr >
			<td >DNA Polymerase I, Large (Klenow) Fragment</td>
			<td >NEB</td>
			<td>M0210S</td>
			<td></td>
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			<td >Klenow Fragment (3&#39;-5&#39; exo)</td>
			<td >NEB</td>
			<td>M0212S</td>
			<td></td>
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			<td >T4 DNA Polymerase</td>
			<td >NEB</td>
			<td>M0203S</td>
			<td></td>
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		<tr >
			<td >T4 Polynucleotide Kinase</td>
			<td >NEB</td>
			<td>M0201S</td>
			<td></td>
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		<tr >
			<td >Deoxynucleotide Solution mix, 10mM</td>
			<td >NEB</td>
			<td>N0447S</td>
			<td></td>
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		<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 ...

Research

JoVE Journal

Genetics

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 ...

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. ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

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) Protocol for Low-abundance Embryonic Samples

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, ...

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

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 ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

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 ...

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 ...

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

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 ...

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...