Bu eser Wayne State Üniversitesi, Karmanos Kanser Enstitüsü, Ulusal Kalp, akciğer ve kan Enstitüsü (1K22HL126842-01A1) B.L.K. için layık bir hibe tarafından desteklenmiştir Bu eser Wayne State Üniversitesi yüksek performans Grid bilgi sayısal kaynaklar için kullanılan (< https://www.grid.wayne.edu/>). Jiji logg CARIP Seq veri analizi ile yardım için teşekkür.
YAZARLARIN KATKILARI:
B.L.K. CARIP-Seq yöntemi gebe, tasarlanmış ve ChIP-Seq ve CARIP-Seq deneyler, sıralama veri analiz ve el yazması askere. Tüm yazarları okudum ve onaylanmış bu el yazması son sürüm.

<ol>
	<li>Barski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129 (4), 823-837 (2007).</li><li>Chen, X., 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=Integration+of+external+signaling+pathways+with+the+core+transcriptional+network+in+embryonic+stem+cells.">Integration of external signaling pathways with the core transcriptional network in embryonic stem cells.</a> Cell. 133 (6), 1106-1117 (2008).</li><li>Boyer, L. 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=Core+transcriptional+regulatory+circuitry+in+human+embryonic+stem+cells.">Core transcriptional regulatory circuitry in human embryonic stem cells.</a> Cell. 122 (6), 947-956 (2005).</li><li>Kim, J., Chu, J., Shen, X., Wang, J., Orkin, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extended+transcriptional+network+for+pluripotency+of+embryonic+stem+cells.">An extended transcriptional network for pluripotency of embryonic stem cells.</a> Cell. 132 (6), 1049-1061 (2008).</li><li>Kidder, B. L., Hu, G., Yu, Z. X., Liu, C., 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=Extended+self-renewal+and+accelerated+reprogramming+in+the+absence+of+Kdm5b.">Extended self-renewal and accelerated reprogramming in the absence of Kdm5b.</a> Mol Cell Biol. 33, 4793-4810 (2013).</li><li>Kidder, B. L., Hu, G., 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=KDM5B+focuses+H3K4+methylation+near+promoters+and+enhancers+during+embryonic+stem+cell+self-renewal+and+differentiation.">KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation.</a> Genome Biol. 15 (2), R32 (2014).</li><li>Zhao, 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=Genome-wide+identification+of+polycomb-associated+RNAs+by+RIP-seq.">Genome-wide identification of polycomb-associated RNAs by RIP-seq.</a> Mol Cell. 40 (6), 939-953 (2010).</li><li>Baltz, A. 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=The+mRNA-bound+proteome+and+its+global+occupancy+profile+on+protein-coding+transcripts.">The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.</a> Mol Cell. 46 (5), 674-690 (2012).</li><li>Castello, 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=Insights+into+RNA+biology+from+an+atlas+of+mammalian+mRNA-binding+proteins.">Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.</a> Cell. 149 (6), 1393-1406 (2012).</li><li>Hudson, W. H., Ortlund, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure,+function+and+evolution+of+proteins+that+bind+DNA+and+RNA.">The structure, function and evolution of proteins that bind DNA and RNA.</a> Nat Rev Mol Cell Bio. 15 (11), 749-760 (2014).</li><li>Khalil, A. 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=Many+human+large+intergenic+noncoding+RNAs+associate+with+chromatin-modifying+complexes+and+affect+gene+expression.">Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.</a> P Natl Acad Sci USA. 106 (28), 11667-11672 (2009).</li><li>Hendrickson, D. G., Kelley, D. R., Tenen, D., Bernstein, B., Rinn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+RNA+binding+by+chromatin-associated+proteins.">Widespread RNA binding by chromatin-associated proteins.</a> Genome Biol. 17, 28 (2016).</li><li>Kelley, R. L., Kuroda, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncoding+RNA+genes+in+dosage+compensation+and+imprinting.">Noncoding RNA genes in dosage compensation and imprinting.</a> Cell. 103 (1), 9-12 (2000).</li><li>Koziol, M. J., Rinn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+traffic+control+of+chromatin+complexes.">RNA traffic control of chromatin complexes.</a> Curr Opin Genet Dev. 20 (2), 142-148 (2010).</li><li>Mercer, T. R., Mattick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+long+noncoding+RNAs+in+epigenetic+regulation.">Structure and function of long noncoding RNAs in epigenetic regulation.</a> Nat Struct Mol Bio. 20 (3), 300-307 (2013).</li><li>Chu, C., 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=Systematic+discovery+of+Xist+RNA+binding+proteins.">Systematic discovery of Xist RNA binding proteins.</a> Cell. 161 (2), 404-416 (2015).</li><li>Mak, W., 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=Reactivation+of+the+paternal+X+chromosome+in+early+mouse+embryos.">Reactivation of the paternal X chromosome in early mouse embryos.</a> Science. 303 (5658), 666-669 (2004).</li><li>Garfield, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+primary+mouse+embryonic+fibroblast+(PMEF)+cultures.">Derivation of primary mouse embryonic fibroblast (PMEF) cultures.</a> Methods Mol Biol. 633, 19-27 (2010).</li><li>Conner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryo+fibroblast+(MEF)+feeder+cell+preparation.">Mouse embryo fibroblast (MEF) feeder cell preparation.</a> Curr Protoc Mol Biol. , (2001).</li><li>Ying, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453 (7194), 519-523 (2008).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nat Methods. 9 (4), 357-359 (2012).</li><li>Zang, C., 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+clustering+approach+for+identification+of+enriched+domains+from+histone+modification+ChIP-Seq+data.">A clustering approach for identification of enriched domains from histone modification ChIP-Seq data.</a> Bioinformatics. 25 (15), 1952-1958 (2009).</li><li>Zhang, Y., 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=Model-based+analysis+of+ChIP-Seq+(MACS).">Model-based analysis of ChIP-Seq (MACS).</a> Genome Biol. 9 (9), R137 (2008).</li><li>Liu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+model-based+Analysis+of+ChIP-Seq+(MACS)+to+analyze+short+reads+generated+by+sequencing+protein-DNA+interactions+in+embryonic+stem+cells.">Use model-based Analysis of ChIP-Seq (MACS) to analyze short reads generated by sequencing protein-DNA interactions in embryonic stem cells.</a> Methods Mol Biol. 1150, 81-95 (2014).</li><li>Kidder, B. L., Hu, G., 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=ChIP-Seq:+technical+considerations+for+obtaining+high-quality+data.">ChIP-Seq: technical considerations for obtaining high-quality data.</a> Nat Immunol. 12 (10), 918-922 (2011).</li><li>Quail, M. 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=A+large+genome+center's+improvements+to+the+Illumina+sequencing+system.">A large genome center's improvements to the Illumina sequencing system.</a> Nat Methods. 5 (12), 1005-1010 (2008).</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></ol>

Yazarlar hiçbir çıkar çatışması beyan ederim.

Çip Seq küresel protein-DNA etkileşimleri konumunu değerlendirmek için yararlı bir yöntemdir (Örn., transkripsiyon faktörleri/histon enzimler/histon modifikasyonları ve DNA değiştirme) ES hücrelerde, yeni geliştirilen CARIP-Seq Protokolü içinde yararlı iken RNA'ların genom çapında Derneği ile kromatin bileşenlerinin sorguluyor. Çip Seq ES Hücre epigenetik manzara ve diğer hücre tipleri değerlendirmek için kullanılan temel bir araçtır. ChIP-Seq ve CARIP-Seq kitaplıkları kalitesini büyük ölçüde ChIP-grade antikorlar üzerinde bağlıdır. ChIP-Seq antikor uygunluğu ChIP-PCR, nerede bir ≥ 3 pozitif kontrol bölgeler kullanılmayan bölgeleri göre 5-fold zenginleştirme için yüksek kaliteli çip Seq veri25oluşturmak için yeterli olmalıdır gerçekleştirerek ampirik olarak belirlenebilir. Aynı şekilde, antikorlar için uygunluk CARIP-Seq için benzer bir şekilde ChIP-Seq için küçük değişiklikler ile açıklandığı gibi test edilebilir. CARIP Protokolü RNA IP adımda yukarıdaki yöntemleri bölümünde açıklandığı gibi takip, Ters-transkripsiyon PCR Q-RT-PCR gerçekleştirmek için şablon olarak kullanılabilir cDNA oluşturmak için yapılmalıdır. Bu çip veya birden fazla genomik bölge, CARIP aşağıdaki zenginleştirme değerlendirmek için tavsiye edilir. Çip ya da CARIP için antikorlar kalitesini değerlendirirken test etmek için ek konuları vardır. Örneğin, ChIP ve CARIP için kullanılan arabellekleri sıkılık NaCl konsantrasyonu değiştirerek optimize: NaCl yoğun antikorlar ile için yararlı olsa da düşük bir NaCl konsantrasyon son derece özel antikorlar için uygundur Non-spesifik bağlama. Yeni nesil sıralama kitaplığı hazırlığı ile ilgili özel astar ve dizinler yerine ticari olarak elde oligonucleotides de kullanılabilir. Ancak, 3' ucunda iki bazlar arasındaki bir phosphorothioate değişiklik de dahil olmak üzere nükleaz sindirim26önlemek için gösterilmiştir olduğunu unutmamak gerekir. Üçüncü taraf RNA-Seq veya çip Seq Kütüphane inşaat kitleri CARIP nın için kitaplıkları oluşturmak ve örnekleri, sırasıyla çip için de kullanılabilir. Ancak, ampirik olarak test kitleri üreten kalite kütüphanelerinde yeni nesil sıralama için onların etkinliğini değerlendirmek için önemlidir. Ek protokoller de ChIP-Seq Kütüphane hazırlık, ChIPmentation27gerçekleştirmek için istihdam edilebilir. Ancak, ChIPmentation uyarlaması burada açıklanan ChIP-Seq protokolüne ek değişiklikler ve daha fazla optimizasyon gerektirir.
Burada, makul sayıda hücreleri (4 × 106 IP başına) kullanarak optimize ChIP ve CARIP iletişim kurallarını açıklar. ChIP protokolü için ara düşük hücre sayıları kullanarak gerkçekleştirilen iken (105 106 hücrelere), CARIP-Seq az 2 × 106 hücreler kullanarak yapmamış. Ancak, çeşitli fiksasyon ve sonication koşullar ampirik olarak test ederek, bu iletişim kuralını kullanarak başarıyla hücreleri az sayıda çalışabilir mümkündür.
Özetle, ChIP-Seq ve CARIP-Seq ES hücrelerde sırasıyla kromatin ilişkili protein ve RNA'ların küresel haritaları oluşturmak için yararlıdır. Bu iletişim kuralları ES hücreler için optimize edilmiş iken, kromatin haritalar çok çeşitli memeli hücre satırları ve doku türleri kullanarak oluşturmak için getirilebilir. Bu protokoller de tek hücreli yonga-Seq veya CARIP-Seq kitaplıkları oluşturmak için optimize.

Embriyonik kök (ES) hücre kader kararlar arasında ekstrasellüler sinyalleri ve transkripsiyon-düzenleyiciler, histon değiştiriciler ve histon kuyrukları sonrası çeviri değiştirilmesi de dahil olmak üzere bir dizi iletişim tarafından düzenlenmektedir. Bu etkileşimler kromatin erişilebilirlik ve kromatin paketlenmesi birine iki durumu kolaylaştırmak: euchromatin, açık ve transcriptionally etkin olan ve sık ve genellikle transcriptionally etkin olmayan heterochromatin. Transkripsiyon faktörleri DNA fingerprinting bağlama benzeşim ve euchromatic bölgeleri ile epigenetik değiştiriciler ortak Gen ifadesinin kontrolünde katılmak için. ChIP-Seq1, dahil olmak üzere yeni nesil sıralama yöntemleri, ES hücre kendini yenileme ve pluripotency2,3için,4 temel genom çapında transkripsiyon ağlar eşleştirmesi'nde vesile olmuştur ,5,6. Ayrıca, RNA immunopreciation yeni nesil (RIP-Seq) sıralama7 değerlendirme RNA-protein etkileşimlerinin tarafından takip ederken DNA bağlayıcı proteinler RNA'ların transkripsiyon olaylar7, düzenleyen ile etkileşim öneririm 8 , 9 , 10 , 11 , 12, kaç çalışmalar kromatin12veya RNA ve histon değişiklikler arasındaki küresel etkileşim ile ilişkili RNA'ların genom çapında lokalizasyonu araştırdı. Uzun kodlamayan RNA'ların (lncRNAs) olan proteinleri kromatin ilişkili13,14,15etkinliği düzenleyen bulduk RNA'ların bir sınıfıdır. Örneğin, Xist düzenleyen, kadın memeli hücrelerinde inactivation bir X kromozomunun, epigenetik repressors16,17alımı yoluyla bir lncRNA olur. Ancak, kromatin ile ilişkili RNA'ların tam spektrum büyük ölçüde bilinmemektedir. Burada, biz yeni bir protokol, kromatin ilişkili RNA immunoprecipitation (CARIP) için yeni nesil sıralama (CARIP-kromatin ilişkili RNA'lar Kütüphane hazırlık dahil olmak üzere ES hücrelerdeki genom-wide olarak tanımlamak için Seq), ardından tarif Yeni nesil sıralama ve histon değişiklikler, transkripsiyon faktörleri ve epigenetik değiştiriciler küresel doluluk eşlemek için ChIP-Seq. Diğer RIP-Seq yöntemleri7farklı olarak, CARIP-Seq kromatin ile ilişkili RNA'ların doğrudan tanımlanması için izin crosslinking ve sonication adımları içerir. Birlikte, ChIP-Seq genom çapında protein-DNA etkileşimleri tanımlamak için güçlü bir araçtır, CARIP-Seq anket için güçlü bir yöntem ise RNA'ların kromatin bileşenleriyle ilişkilendirilmiş.

<table><tbody><tr><td>Mouse leukemia inhibitory factor (ESGRO LIF)</td><td>EMD Millipore</td><td></td><td>10&lt;sup&gt;6 &lt;/sup&gt;or 10&lt;sup&gt;7 &lt;/sup&gt;units</td></tr><tr><td>GSK3&amp;beta; inhibitor CHIR99021 (GSK3i)</td><td>Stemgent</td><td></td><td>Solvent: DMSO 10mM</td></tr><tr><td>MEK inhibitor PD0325901 (MEKi)</td><td>Stemgent</td><td></td><td>Solvent: DMSO 10mM</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM), high glucose</td><td>Invitrogen</td><td>11965092</td><td></td></tr><tr><td>PBS (phosphate buffered saline)</td><td>Invitrogen</td><td>10010031</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>2-Mercaptoethanol (50 mM)</td><td>Gibco</td><td>31350010</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>Gibco</td><td>11140076</td><td></td></tr><tr><td>EmbryoMax ES Cell Qualified Fetal Bovine Serum, 500 ml</td><td>EMD Millipore</td><td>ES-009-B</td><td></td></tr><tr><td>EmbryoMax 0.1% Gelatin Solution</td><td>EMD Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7126-500G</td><td>1.25 M stock conentration in water</td></tr><tr><td>Formaldehyde solution</td><td>Sigma</td><td>F8775-500ML</td><td></td></tr><tr><td>TE (Tris EDTA) pH 8.0 1X</td><td>Quality Biological</td><td>351-011-131</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF) solution</td><td>Sigma</td><td>93482-250ML-F</td><td></td></tr><tr><td>cOmplete Protease Inhibitor Cocktail</td><td>Roche</td><td>11697498001</td><td></td></tr><tr><td>Sodium dodecyl sulfate solution (20%)</td><td>Quality Biological</td><td>A611-0837-10</td><td></td></tr><tr><td>Q125 sonifier</td><td>Qsonica</td><td>4422</td><td></td></tr><tr><td>Triton X-100 solution</td><td>Sigma</td><td>93443-100ML</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>30970</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Dynabeads Protein G for Immunoprecipitation</td><td>Invitrogen</td><td>10004D</td><td></td></tr><tr><td>Dynabeads Protein A for Immunoprecipitation</td><td>Invitrogen</td><td>10002D</td><td></td></tr><tr><td>Dynabeads Protein A/Protein G and Magnet Starter Pack</td><td>Invitrogen</td><td>10015D</td><td></td></tr><tr><td>Lithium chloride</td><td>Sigma</td><td>L4408</td><td></td></tr><tr><td>IGEPAL CA-630</td><td>Sigma</td><td>I8896</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Dynabeads mRNA Purification Kit (for mRNA purification from total RNA preps)</td><td>Invitrogen</td><td>61006</td><td></td></tr><tr><td>SuperScript Double-Stranded cDNA Synthesis Kit</td><td>Invitrogen</td><td>11917010</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>End-It DNA End-Repair Kit</td><td>Lucigen</td><td>ER81050</td><td></td></tr><tr><td>Klenow Fragment (3&#039;&amp;rarr;5&#039; exo-)</td><td>NEB</td><td>M0212L</td><td></td></tr><tr><td>dATP (10 mM)</td><td>Invitrogen</td><td>18252015</td><td></td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202L</td><td></td></tr><tr><td>TrackIt 1 Kb Plus DNA Ladder</td><td>Invitrogen</td><td>10488085</td><td></td></tr><tr><td>E-gel EX agarose gels, 2%</td><td>Invitrogen</td><td>G402002</td><td></td></tr><tr><td>Phusion high-fidelity 2X master mix with HF buffer</td><td>Thermo Fisher</td><td>F531LPM</td><td></td></tr><tr><td>ChIPAb+ Trimethyl-Histone H3 (Lys4) - ChIP Validated Antibody</td><td>EMD Millipore</td><td>17-614</td><td></td></tr><tr><td>Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)</td><td>Abcam</td><td>ab32356</td><td></td></tr><tr><td>Corning Costar Flat Bottom Cell Culture Plates (6-well)</td><td>Fisher</td><td>720083</td><td></td></tr><tr><td>Falcon Standard Tissue Culture Dishes (10cm)</td><td>Fisher</td><td>08772E</td><td></td></tr><tr><td>Bioruptor pico</td><td>Diagenode</td><td>B01010001</td><td></td></tr><tr><td>Qubit Flurometer 2.0</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher</td><td>Q32851</td><td></td></tr><tr><td>MinElute Reaction Cleanup Kit</td><td>Qiagen</td><td>28204</td><td></td></tr><tr><td>MinElute Gel Extraction Kit</td><td>Qiagen</td><td>28604</td><td></td></tr><tr><td>Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)</td><td>Abcam</td><td>ab9053</td><td></td></tr><tr><td>Labquake rotator</td><td>Thermo Fisher</td><td>400110Q</td><td></td></tr><tr><td>Illumina HiSeq platform</td><td>Illumina</td><td></td><td></td></tr><tr><td>TURBO DNase</td><td>Invitrogen</td><td>AM2238</td><td></td></tr></tbody></table>

carip-seq and chip-seq: methods to identify chromatin-associated rnas and protein-dna interactions in embryonic stem cells

Embriyonik kök (ES) hücre kendini yenileme ve farklılaşma dışsal sinyalleri ve transkripsiyon faktörleri, epigenetik düzenleyiciler ve combinatorially gen etkisi histon sonrası çeviri değişiklikler iç ağlar tarafından yönetilir Yakındaki genlerin ifade durumu. Kromatin dinamikleri ve gen ekspresyonu düzenleyen çeşitli proteinler ile etkileşim için RNA da gösterilmiştir. Kromatin ilişkili RNA immunoprecipitation (CARIP) yeni nesil sıralama (CARIP-Seq) tarafından takip RNA'ların kromatin proteinleri kromatin immunoprecipitation ardından yeni nesil sıralama () tarafından süre ile ilgili ankete roman bir yöntemdir ChIP-Seq) histon, transkripsiyon faktörleri ve küresel ölçekte-ES hücrelerdeki epigenetik değiştiriciler translasyonel modifikasyon konumunu eşlemek için bir güçlü genomik tekniktir. Burada, CARIP-Seq ve ChIP-Seq yeni nesil sekanslama için Kütüphane inşaat dahil olmak üzere, küresel kromatin ilişkili RNA ve epigenomic haritalar ES hücreleri oluşturmak için gerçekleştirmek için yöntemleri açıklanmaktadır.

Başarılı bir şekilde H3K4me3, H3K4me2 ve KDM5B bu çip Seq Protokolü6kullanarak ES hücrelerdeki genom çapında bağlama sorguladık. ES hücreler kültürlü besleyici-Alerjik koşullarda (Şekil 1) ve yukarıda açıklandığı gibi çapraz bağlı. Sonication daha sonra ChIP-Seq Protokolü 3.2. adımında açıklandığı gibi gerçekleştirilen ve DNA üzerinde bir % 2 özel jel (Şekil 2) çalıştırarak değerlendirilir. Daha sonra ChIP adım 4'te açıklandığı gibi gerçekleştirildi ve Kütüphane hazırlık 6. adımda açıklandığı gibi gerçekleştirildi. Çip Seq kitaplıkları bir nesil sıralama platformunda sıralı. Temsilcisi özel UCSC tarayıcı görünümler zenginleştirme çekirdek pluripotency gene POU5F1 (Şekil 3A) ve HOXC küme (Şekil 3B) H3K4me3, H3K4me2 ve KDM5B ortaya koyuyor. CARIP-Seq da 4-6 adımda açıklandığı gibi protokol sonrası tarafından gerçekleştirildi. Bir temsilci UCSC tarayıcı görünümü bir loci ES hücrelerdeki (Şekil 4), H4K20me3 ilişkili RNA (CARIP-Seq) ve H4K20me3 doluluk (ChIP-Seq) zenginleştirme ortaya koymaktadır. CARIP-Seq sinyal RNA H4K20me3 ile ilişkili olmakla birlikte, bu gösterilen odağı ile etkileşim gelmeyebilir olduğunu gösterir. CARIP-Seq veri kümeleri benzer bir şekilde ChIP-Seq veri kümeleri analiz edilebilir. Fare ES oluşturulan veri hücre CARIP-Seq ve ChIP-Seq deneyler her ikisi de uyumlu olabilir bir başvuru genom (Örn., mm9, mm10) kullanarak bowtie221. Çip Seq ve CARIP-Seq zenginleştirilmiş bölgeler her ikisi de "Kayma kümeleme için kimlik ChIP-Enriched bölgeler" (SICER)22 veya MAC'ler23,24gibi tepe arama programları kullanılarak belirlenebilir. CARIP-Seq veri kümeleri büyük olasılıkla geniş doruklarına içerdiğinden, geniş ve keskin doruklarına tanımlayabilirsiniz bir algoritma kullanmak önemlidir. Bu el yazması açıklanan protokoller ChIP-Seq ve CARIP-Seq gerçekleştirmek için optimize edilmiş iken ES hücreleri kullanarak, bu protokollerin de protein-DNA ve RNA etkileşimlerde kromatin ilişkili diğer hücre tipleri değerlendirmek için uygun olmalıdır.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57481/57481fig1.jpg"> Şekil 1: besleyici-Alerjik kültür ES hücre. (A) deneysel tasarım. (B) ES Hücre ES iMEFs kültürlü LIF içeren medya hücre veya koşullarda besleyici-Alerjik ES ile LIF ve (C) GSK3i veya (D) GSK3i ve MEKi (2i) içeren medya hücre. <a href="https://cloudflare.jove.com/files/ftp_upload/57481/57481fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57481/57481fig2.jpg"> Şekil 2: çapraz ES Hücre kromatin Sonication. Çapraz ES hücreleri sonicated bir sonifier bir microtip ile 18 devredir kullanarak (% 40 genlik, 30 s ve 30 s dinlenme). Crosslinking tersine ve RNase sindirim daha sonra RNA kaldırmak için gerçekleştirildi. DNA PCI kullanarak ve üzerinde bir özel jel (% 2) çalıştırın saflaştırıldı. <a href="https://cloudflare.jove.com/files/ftp_upload/57481/57481fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57481/57481fig3.jpg"> Resim 3: bir histon enzim ve histon değişiklikleri ES hücrelerdeki değiştirme analizini temsilcisi ChIP-Seq. Özel UCSC tarayıcı görünümlerini H3K4me3, H3K4me2 ve KDM5B pluripotency-Regülatör (A) POU5F1 ve (B) HOXC küme ES hücrelerdeki (normalleştirilmiş etiketi yoğunluğu (RPBM), Bankası milyon okuma başına başına okuyun). KDM5B bağlama ve H3K4me3/2 doluluk arasında örtüşme unutmayın. <a href="https://cloudflare.jove.com/files/ftp_upload/57481/57481fig3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57481/57481fig4.jpg"> Şekil 4: RNA'ın temsilcisi CARIP-Seq analiz ilişkili bir histon değişiklik ve ChIP-Seq ES hücrelerdeki. UCSC tarayıcı sayısı H4K20me3 CARIP-Seq ve H4K20me3 ChIP-Seq sinyallerinin ES hücrelerdeki (normalleştirilmiş etiketi yoğunluğu (RPBM), Bankası milyon okuma başına başına okuyun). <a href="https://cloudflare.jove.com/files/ftp_upload/57481/57481fig4large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

Watch this Scientific Journal Video about CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells at JoVE.com

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Burada, biz ChIP-Seq gerçekleştirmek için yöntemleri tarif ve CARIP-yeni nesil sıralama, Kütüphane hazırlık dahil olmak üzere küresel epigenomic ve kromatin ilişkili RNA oluşturmak için Seq, ES hücrelerde eşler.

CARIP-Seq ve ChIP-Seq: kromatin ilişkili RNA ve Protein-DNA etkileşimleri embriyonik kök hücreleri tanımlamak için yöntemleri

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

carip-seq-chip-seq-methods-to-identify-chromatin-associated-rnas

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.8K Görüntüleme.  Wayne State University School of Medicine. Burada, biz ChIP-Seq gerçekleştirmek için yöntemleri tarif ve CARIP-yeni nesil sıralama, Kütüphane hazırlık dahil olmak üzere küresel epigenomic ve kromatin ilişkili RNA oluşturmak için Seq, ES hücrelerde eşler.

Video: CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

Kromatin Regülatörleri ve Devletleri&#39;nde genom Anlık<em&gt; Ksenopus</emChIP-Sıra tarafından&gt; Embriyolar

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Gelişim Biyolojisi

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

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

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

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

ChEC-seq ile Protein-DNA Etkileşimlerinin Genom Boyunca Haritalandırılması<em&gt; Saccharomyces cerevisiae</em

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

Genetik

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.

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, ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Embriyonik Kök Hücre ve Doku Kardiyak Farklılaşma epigenetik Yönetmelik

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Tahmin ve Gen Düzenleme Elementlerin Doğrulama Retinoik Asit Kaynaklı Embriyonik sırasında Aktif Hücre Farklılaşma Kök

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Biyoloji

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo) Kullanılarak Fare Kök Hücre Türevi Nöronlarda Protein-DNA Etkileşimlerinin Yüksek Çözünürlüklü Haritalanması

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Erken evre Fare Embriyolar kullanarak Doku özel Genler Kromatin Immunoprecipitation Testi

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA Saflaştırma tarafından Kromatin İzolasyon (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Yüksek verim Sıralama için Yüksek Kalite Kromatin Immunoprecipitation DNA Şablon üretimi (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

CARIP-Seq ve ChIP-Seq: kromatin ilişkili RNA ve Protein-DNA etkileşimleri embriyonik kök hücreleri tanımlamak için yöntemleri

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