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>
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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>106 or 107 units</td>
		</tr>
		<tr>
			<td>GSK3&#946; 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&#39;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&#39;&#8594;5&#39; 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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

9.7K 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

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Serum serbest Tek tabakalı Kültüründe Fare Embriyonik Kök Hücre Sinir Farklılaşma

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Yaratma CRISPR / Cas9 aracılığı monoallelik Delesyonlar Fare Embriyonik Kök Hücreleri Artırıcı Fonksiyonu Eğitim için

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

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 ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Fare Embriyonik Hemogenic endotel hücrelerinin izolasyonu

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Embriyonik Kök Hücre Pluripotansını Yüksek Verimli İnhibitör Taramayla Düzenleyen Kinazları Tanımlamak İçin Basit Bir Yöntem

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Yatay Dağ Dağı: Derinin kalın, üç boyutlu doku kesitleri için yeni bir İşleme ve Görüntüleme Protokolü

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Hızlı, yönlendirilmiş farklılaşma insan embriyonik veya İndüklenmiş Pluripotent kök hücrelerinden retina Pigment epitel hücrelerinin

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Yaşam sağlam Zebra balığı tek hücreli Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Çekirdeklerin dağıtım, proteinler ve sitoiskeleti çalışmaya Caenorhabditis elegans Germline sayısal analizi

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...