Yazarlar Jan Appel bu protokolü kurulması sırasında onun mükemmel teknik destek için teşekkür ederiz. Rada-Iglesias laboratuvar çalışmalarında intramural CMMC tarafından desteklenmektedir finansman, DFG araştırma hibe (RA 2547/1-1, RA 2547/2-1 ve TE 1007/3-1) UoC gelişmiş araştırmacı grup Grant ve CECAD Grant.

<ol>
	<li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293 (5532), 1074-1080 (2001).</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=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128 (4), 693-705 (2007).</li><li>Wang, Z., Schones, D. E., 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=Characterization+of+human+epigenomes.">Characterization of human epigenomes.</a> Curr Opin. Genet Dev. 19 (2), 127-134 (2009).</li><li>ENCODE Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Roadmap Epigenomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+111+reference+human+epigenomes.">Integrative analysis of 111 reference human epigenomes.</a> Nature. 518 (7539), 317-330 (2015).</li><li>Heintzman, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+modifications+at+human+enhancers+reflect+global+cell-type-specific+gene+expression.">Histone modifications at human enhancers reflect global cell-type-specific gene expression.</a> Nature. 459 (7243), 108-112 (2009).</li><li>Rada-Iglesias, A., Bajpai, R., Swigut, T., Brugmann, S. A., Flynn, R. A., Wysocka, 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+unique+chromatin+signature+uncovers+early+developmental+enhancers+in+humans.">A unique chromatin signature uncovers early developmental enhancers in humans.</a> Nature. 470 (7333), 279-283 (2011).</li><li>Creyghton, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H3K27ac+separates+active+from+poised+enhancers+and+predicts+developmental+state.">Histone H3K27ac separates active from poised enhancers and predicts developmental state.</a> Proc Nat Acad Sci USA. 107 (50), 21931-21936 (2010).</li><li>Benayoun, B. 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=H3K4me3+breadth+is+linked+to+cell+identity+and+transcriptional+consistency.">H3K4me3 breadth is linked to cell identity and transcriptional consistency.</a> Cell. 158 (3), 673-688 (2014).</li><li>Rehimi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenomics-Based+Identification+of+Major+Cell+Identity+Regulators+within+Heterogeneous+Cell+Populations.">Epigenomics-Based Identification of Major Cell Identity Regulators within Heterogeneous Cell Populations.</a> Cell Rep. 17 (11), 3062-3076 (2016).</li><li>Whyte, W. 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=Master+transcription+factors+and+mediator+establish+super-enhancers+at+key+cell+identity+genes.">Master transcription factors and mediator establish super-enhancers at key cell identity genes.</a> Cell. 153 (2), 307-319 (2013).</li><li>Valouev, 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=Genome-wide+analysis+of+transcription+factor+binding+sites+based+on+ChIP-Seq+data.">Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data.</a> Nat Methods. 5 (9), 829-834 (2008).</li><li>Dahl, J. 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=Broad+histone+H3K4me3+domains+in+mouse+oocytes+modulate+maternal-to-zygotic+transition.">Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.</a> Nature. 537 (7621), 548-552 (2016).</li><li>Rotem, 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=Single-cell+ChIP-seq+reveals+cell+subpopulations+defined+by+chromatin+state.">Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state.</a> Nat Biotech. 33 (11), 1165-1172 (2015).</li><li>Schmidl, C., Rendeiro, A. F., Sheffield, N. C., Bock, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIPmentation:+fast,+robust,+low-input+ChIP-seq+for+histones+and+transcription+factors.">ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors.</a> Nat Methods. 12 (10), 963-965 (2015).</li><li>Zhang, 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=Allelic+reprogramming+of+the+histone+modification+H3K4me3+in+early+mammalian+development.">Allelic reprogramming of the histone modification H3K4me3 in early mammalian development.</a> Nature. 537 (7621), 553-557 (2016).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev Dynam. 195 (4), 231-272 (1951).</li><li>Ho, J. W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+metazoan+chromatin+organization.">Comparative analysis of metazoan chromatin organization.</a> Nature. 512 (7515), 449-452 (2014).</li><li>Calo, E., Wysocka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+enhancer+chromatin:+what,+how,+and+why?.">Modification of enhancer chromatin: what, how, and why?.</a> Mol Cell. 49 (5), 825-837 (2013).</li><li>Spitz, F., Furlong, E. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors:+from+enhancer+binding+to+developmental+control.">Transcription factors: from enhancer binding to developmental control.</a> Nat Rev Genetics. 13 (9), 613-626 (2012).</li></ol>

Yazarlar herhangi bir rakip mali çıkarlarının açıklanması gerekir var mı.

Epigenomic çip seq kullanarak Histon modifikasyonu profil oluşturma farklı hücresel bağlamlarda4,5,18omurgalı genleri fonksiyonel ek açıklama geliştirmek için kullanılabilir. Bu epigenomic profilleri, diğer şeyler arasında artırıcı öğeleri, arttırıcılar (hazır veya hazırYani, aktif,) yasal durumunu tanımlamak ve farklı biyolojik büyük hücre kimlik düzenleyiciler tanımlamak için tanımlama...

Histon translasyonel modifikasyonlar doğrudan transkripsiyon, çoğaltma ve DNA onarım1,2,3de dahil olmak üzere çeşitli Kromatin bağlı işlemlerde söz konusu. Ayrıca, farklı Histon değişiklikleri olumlu gösterir (Örneğin, H3K4me3 ve H3K27ac) veya negatif (Örneğin, H3K9me3 ve H3K27me3) gen ekspresyonu ile korelasyon ve genel olarak tanımlanabilir etkinleştirebilir veya baskıcı Histon işaretler gibi sırasıyla2,3. Sonuç olarak, küresel Histon değişiklik haritalar, epigenomic haritalar, olarak da işlevsel olarak omurgalı genleri4,5ek açıklama eklemek için güçlü ve evrensel araçlar ortaya çıkmıştır. Arttırıcılar tespit edilmesi gibi örneğin, distal düzenleyici dizileri belirli Kromatin imzalar varlığı dayalı (Örneğin, etkin arttırıcılar: H3K4me1 ve H3K27ac), hangi proksimal organizatörü bölgelerden onları ayırmak (Örneğin, etkin rehberleri: H3K4me3)6,7,8. Öte yandan, genlerin büyük hücre kimlik düzenleyici fonksiyonları ile genellikle H3K4me3 H3K27me3, altta yatan genlerin transcriptionally etkin veya etkin olmayan durumuna bağlı olarak sırasıyla yine geniş Kromatin etki alanları ile9 bulunur ,10. Benzer şekilde, büyük hücre kimlik genlerin ifade sık sık çoklu tarafından ve dağınık şekilde kontrol edilebilir için geniş H3K27ac işaretlenmiş etki alanları11tanımlanabilir arttırıcılar (Yani, süper arttırıcılar), kümelenmiş gibi görünüyor.
Şu anda, Histon değişiklik haritalar sağlayan ChIP-yonga (ChIP microarrays için birleştiğinde) gibi önceki yaklaşımlar ile karşılaştırıldığında daha yüksek çözünürlük, daha az eserler, daha az gürültü, daha fazla kapsama alanı ve alt ChIP-seq teknolojisi kullanılarak oluşturulan maliyeti12. Yine de, ChIP-seq teknolojisini kullanarak epigenomic haritalar nesil çoğunlukla çip faiz örneklerinde başarılı bir şekilde gerçekleştirmek için kapasite ile ilişkili doğasında kendi sınırlamaları vardır. Geleneksel çip protokolleri genellikle milyonlarca hücre hangi sınırı içinde in vitro hücre için bu yöntemin uygulanabilirliği hatları veya bu hücreler kolayca izole vivo içinde (Örneğin, kan hücreleri) olabilir, gerekli. Son birkaç yıl içinde bir dizi modifiye ChIP Protokolü düşük hücre sayıları ile uyumlu açıklanan13,14,15,16olmuştur. Ancak, bu iletişim kuralları (Yani, ChIP-seq) yeni nesil sıralama ile birleştiğinde olabilir için özel olarak tasarlanmış ve genellikle özel Kütüphane hazırlama yöntemleri13,14, kullanın 15 , 16.
Burada, Histon değişiklik profilleri için ara düşük hücre sayıları (5 x 104 - 5 x 105 hücreleri) kullanarak araştırmak için kullanılan bir çip protokol açıklanan ya da seçili loci (Yani, ChIP-qPCR) veya genel olarak (Yani, Çip seq) (şekil 1). ChIP-seq teknoloji birleştiğinde, bizim çip Protokolü standart kitaplığı hazırlama yöntemleri, böylece birçok laboratuar10genel olarak erişilebilir yapmak ile birlikte kullanılabilir. Bu iletişim kuralı birkaç Histon işaretleri araştırmak için kullanılan (örneğin, H3K4me3, H3K27me3 ve H3K27ac) farklı tavuk embriyonik dokularda (Örneğin, spinal Nöral tüp (SNT), iyi çikinti ve epiblast). Ancak, biz genel olarak hangi biyolojik ve/veya klinik olarak ilgili örnekleri yalnızca düşük miktarlarda elde edilebilir diğer organizmalar için uygulanabilir olmalıdır tahmin.

<table>
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA powder</td>
			<td>Carl Roth</td>
			<td>3737.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Saline buffer (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL pH8.0</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>3957.2</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-Deoxycholate</td>
			<td>Sigma Aldrich</td>
			<td>D6750-24</td>
			<td></td>
		</tr>
		<tr>
			<td>N-lauroylsarcosine</td>
			<td>Sigma Aldrich</td>
			<td>61743-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Applichem</td>
			<td>A3724,0250</td>
			<td></td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Carl Roth</td>
			<td>3739.2</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma Aldrich</td>
			<td>I3021-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Carl Roth</td>
			<td>1833</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G/magnetic beads</td>
			<td>Invitrogen</td>
			<td>1004D</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>252549-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNase</td>
			<td>Peqlab</td>
			<td>12-RA-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma Aldrich</td>
			<td>46.35 E</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-butyrate</td>
			<td>Sigma Aldrich</td>
			<td>SLB2659V</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase inhibitor</td>
			<td>Roche</td>
			<td>5892791001</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBRgreen Mix</td>
			<td>biozym</td>
			<td>617004</td>
			<td></td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>Sigma Aldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>1Kb ladder</td>
			<td>Thermofisher</td>
			<td>SM1333</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Sigma Alrich</td>
			<td>03756-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA (1X)</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Octylphenol Ethoxylate (Triton X 100)</td>
			<td>Roth</td>
			<td>3051.4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#180;s Modified Eagle Medium)</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel loading tips Multiflex</td>
			<td>A.Hartenstein</td>
			<td>GS21</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR Plates</td>
			<td>Sarstedt</td>
			<td>721,985,202</td>
			<td></td>
		</tr>
		<tr>
			<td>384 well</td>
			<td>Sarstedt</td>
			<td>721,985,202</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Sarstedt</td>
			<td>72,706</td>
			<td></td>
		</tr>
		<tr>
			<td>100-1000&#181;l Filter tips</td>
			<td>Sarstedt</td>
			<td>70,762,211</td>
			<td></td>
		</tr>
		<tr>
			<td>2-20&#181;l Filter tips</td>
			<td>Sarstedt</td>
			<td>70,760,213</td>
			<td></td>
		</tr>
		<tr>
			<td>2-200&#181;l Filter tips</td>
			<td>Sarstedt</td>
			<td>70,760,211</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5-10&#181;l Filter tips</td>
			<td>Sarstedt</td>
			<td>701,116,210</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K27me3 antibody</td>
			<td>Active Motif</td>
			<td>39155</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K27ac antibody</td>
			<td>Active Motif</td>
			<td>39133</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K4me3 antibody</td>
			<td>Active Motif</td>
			<td>39159</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K4me2 antibody</td>
			<td>Active Motif</td>
			<td>39141</td>
			<td></td>
		</tr>
		<tr>
			<td>End Repair Mix</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Paramagnetic beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td></td>
		</tr>
		<tr>
			<td>Resuspension buffer</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>A-Tailing Mix</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ligation Mix</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Adapter Indexes</td>
			<td>Illumina</td>
			<td>RS-122-2101</td>
			<td></td>
		</tr>
		<tr>
			<td>Stop Ligation Buffer</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Primer Cocktail</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced PCR Mix</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA ladder</td>
			<td>Agilent</td>
			<td>5067-5582</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution Buffer</td>
			<td>Agilent</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Buffer</td>
			<td>Agilent</td>
			<td>5067-5582</td>
			<td></td>
		</tr>
		<tr>
			<td>Library Quantification Kit</td>
			<td>Kapa Biosystems</td>
			<td>KK4835 &#8211; 07960204001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertile chicken white eggs</td>
			<td>LSL Rhein Main</td>
			<td>KN: 15968</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle (Neoject)</td>
			<td>A.Hartenstein</td>
			<td>10001</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (Ecoject 10ml)</td>
			<td>Dispomed witt oHG</td>
			<td>21010</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hermle</td>
			<td>Z 216 MK</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoshaker</td>
			<td>ITABIS</td>
			<td>MKR13</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Active Motive</td>
			<td>EpiShear probe sonicator</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Diagenode</td>
			<td>Bioruptor Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Stuart</td>
			<td>SB3</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable volume (5&#8211;1,000 &#956;l) pipettes</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Sigma</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic holder</td>
			<td>Thermo Fisher</td>
			<td>DynaMag-2 12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Table spinner</td>
			<td>Heathrow Scientific</td>
			<td>Sprout</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer</td>
			<td>LMS</td>
			<td>VTX 3000L</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-Time PCR Cycler</td>
			<td>Roche</td>
			<td>Light Cycler; Serial Nr.5662</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Cycler</td>
			<td>Applied Biosystems</td>
			<td>Gene Amp PCR System 9700</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA and RNA quality control system</td>
			<td>Agilent</td>
			<td>Agilent 4200 TapeStation System</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>5-Inox-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Perforated spoon</td>
			<td>World precision instruments</td>
			<td>501997</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation (chip) protocol for low-abundance embryonic samples

Kromatin immunoprecipitation (ChIP) post-translationally tarihinde Histon, Histon çeşitleri, transkripsiyon faktörleri ya da Kromatin değiştirici enzimler belirli bir odağı veya bir genom geniş ölçekte lokalizasyonu eşleştirmek için bir yaygın kullanılan tekniktir. ChIP deneyleri ile yeni nesil sıralama (Yani, ChIP-Seq) kombinasyonu genel olarak gen düzenleyici ağları ortaya çıkarmak için fonksiyonel ek açıklama genleri, özellikle sıralarının kodlamayan düzenleyici geliştirmek için güçlü bir yaklaşımdır. Çip iletişim kuralları normalde böylece nadir hücre tipleri veya küçük doku biyopsisi soruşturma için bu yöntem uygulanabilirliği iyileştirmelerden hücresel malzeme büyük miktarda gerektirir. Biz burada hangi adımları tamamlamak için gereken basitleştirilmiş bir çip Protokolü çip tahlil genellikle vivo içinde sırasında erken omurgalı embriyo elde edilebilir biyolojik malzeme miktarı ile uyumlu hale getirmek için tarif tahlil örnek kaybı en aza indirmek için azaltılmış. Bu çip protokolü farklı Histon değişiklikler çeşitli embriyonik tavuk ve yetişkin fare dokular Orta hücre sayıları (5 x 104 - 5 x 105 hücreleri) düşük kullanarak araştırmak için başarılı bir şekilde kullanılmıştır. Önemlisi, bu iletişim kuralı standart kitaplığı hazırlama yöntemleri kullanarak ChIP-seq teknoloji ile uyumludur, böylece küresel epigenomic sağlayan son derece alakalı embriyonik dokularda haritalar.

Bizim çip protokol performansını göstermek için biz ChIP-seq HH19 tavuk bebekleri havuza alınan SNT bölümleri kullanarak deneyler, HH22 üst çıkıntıların tavuk embriyo ve sahne-HH3 tavuk bebekleri bağlama profilleri araştırmak için gerçekleştirilen çeşitli Histon değişiklikler (Yani, H3K4me2, H3K27ac, H3K4me3 ve H3K27me3). Bir kez çip DNA'lar elde edilmiştir, sonication verimliliği karşılık gelen giriş DNA'lar (şekil 3)...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples at JoVE.com

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Burada, Kromatin immunoprecipitation (ChIP) ve ChIP-seq Kütüphane hazırlık Protokolü düşük-bereket tavuk embriyonik örnekleri küresel epigenomic profilleri oluşturmak için açıklar.

Kromatin Immunoprecipitation (ChIP) protokolü için düşük-bereket embriyonik örnekleri

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

chromatin-immunoprecipitation-chip-protocol-for-low-abundance

Research

JoVE Journal

Genetics

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

15.6K Görüntüleme.  University of Cologne. Burada, Kromatin immunoprecipitation (ChIP) ve ChIP-seq Kütüphane hazırlık Protokolü düşük-bereket tavuk embriyonik örnekleri küresel epigenomic profilleri oluşturmak için açıklar.

Video: Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

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.

Kromatin kompleksleri değiştirme özelliklerini bağlama toplu Kromatin analizi için sıralı tuz çekimi

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

Genetik

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.

Kromatin İmmunopresipitasyon Yöntemi Kullanılarak c-KIT Ligand Promotör Analizi

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.

Fare T-hücresi Hatlarında Kromatin İmmünopresipitasyonu (ChIP)

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.

Nucleosome yoğunluk analiz için kitaplıklarına sıralama yerli Kromatin Immunoprecipitation nesil

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

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.

Histon değişiklikler Saccharomyces cerevisiae üzerinden Kromatin Immunoprecipitation (ChIP)

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

CRISPR-Mediated Reorganization of Chromatin Loop Structure

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

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

Kromatin döngü yapısı yeniden yapılanma CRISPR-aracılı

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

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

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

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

Kromatin Immunoprecipitation fare kahverengi yağ dokusunun

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.

Mammalin hücrelerinde Micrococcal Nüklenleri kullanarak kromatin Immünopmamitasyon tahlil

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

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

Kromatin Etki Alanlarının De novo Oluşumunu İnceleme Yöntemi

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

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.

Kromatin İmmünenasyon ve Yüksek İş Kelejisi Kullanarak Salmonella Biyofilmde CsgD Düzenleyici Hedeflerin Keşfedilmesi (ChIP-seq)

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