Name: carip-seq and chip-seq: methods to identify chromatin-associated rnas and protein-dna interactions in embryonic stem cells
Uploaded: 2018-05-25T14:00:00
Description: 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

这项工作得到了韦恩州立大学 Karmanos 癌症研究所的支持, 由国家心脏、肺和血液研究所 (1K22HL126842-01A1) 授予 B.L.K。这项工作利用韦恩州立大学高性能计算网格计算资源 (< https://www.grid.wayne.edu/>)。我们感谢集集 Kurup 对 CARIP 数据分析的帮助。
作者的贡献:
B.L.K. 构思了 CARIP 法, 设计并进行了芯片序列和 CARIP 的实验, 分析了测序数据, 起草了手稿。所有作者都已阅读并批准了本手稿的最终版本。

1. 小鼠 ES 细胞在无饲养条件下的培养。
注意: 小鼠 ES 细胞是传统的在培养基上培养的细胞培养皿上涂有明胶和单层小鼠胚胎成纤维细胞 (MEF), 已 mitotically 灭活 (iMEFs)。然而, MEFs 应在下游表观或表达分析之前被删除, 以防止与 MEF 相关的染色质和 RNA 的污染。
<ol><li>给料层的制备: 在水中加入2毫升0.1% 的明胶, 并在37摄氏度下孵育20-30 分钟, 明胶涂层 a 6 井细胞培养板。</li>
	<li>?...

<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>

在 ES 细胞中, 芯片序列是评价全球蛋白质-DNA 相互作用 (e. g)、转录因子/组蛋白修饰酶/组蛋白修饰和 DNA 的有效方法, 而新开发的 CARIP 序列协议在审问基因组范围内的 rna 与染色质成分的关联。芯片序列是一种基本的工具, 用于评估 ES 细胞和其他细胞类型的表观景观。芯片序列和 CARIP 库的质量在很大程度上依赖于芯片级抗体。对芯片序列的抗体的适用性可以通过执行芯片 PCR 的经验来确定, ?...

胚胎干细胞的命运决定由细胞外信号和一系列转录调控器之间的通讯调节, 其中包括组蛋白修饰语和组蛋白尾的翻译后修饰。这些相互作用促进染色质可达性和染色质的包装成两种状态之一: 染色质, 是开放和转录活跃的, 染色质, 这是紧凑的, 一般转录不活跃。转录因子与 DNA 序列特异的结合亲和性和表观遗传修饰, euchromatic 区域参与控制基因表达。下一代测序方法, 包括芯片序列1, 在映射全基因组转录网络 (ES 细胞自我更新和干细胞2、3、4) 方面发挥了重要作用. ,5,6。此外, 虽然 rna immunopreciation 其次是下一代测序 (RIP 序列)7对 RNA-蛋白质相互作用的评价表明, DNA 结合蛋白与 rna 相互作用来调节转录事件7,8,9,10,11,12, 很少有研究调查了与染色质12相关的 rna 的全基因组定位, 或者 rna 和组蛋白修饰之间的全球相互作用。长非编码 rna (lncRNAs) 是一种 rna, 它已被发现来调节染色质相关蛋白的活性13,14,15。例如, Xist 是一种 lncRNA, 通过招募阻遏16、17, 在雌性哺乳动物细胞中调节一个 X 染色体的失活。然而, 与染色质相关的 rna 的全谱在很大程度上是未知的。在这里, 我们描述了一个新的协议, 染色质相关的 RNA 免疫沉淀 (CARIP) 其次是下一代测序 (CARIP 序列), 以确定染色质相关 rna 在全基因组的基础上的 ES 细胞, 包括图书馆准备下一代测序, 和芯片序列, 以映射全球占用的组蛋白修饰, 转录因子, 和表观遗传修饰。与其他 RIP 序列方法7不同, CARIP 序列包括交联和超声波步骤, 它允许直接识别与染色质相关的 rna。在一起, 芯片序列是一个强有力的工具, 以确定全基因组范围内的蛋白质-DNA 相互作用, 而 CARIP 是一个强大的方法来调查与染色质成分相关的 rna。

<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

胚胎干细胞的自我更新和分化受外部信号和转录因子的内在网络、表观遗传调控、combinatorially 对基因组蛋白的转换后修饰的制约。附近基因的表达状态。RNA 也被证明与各种各样的蛋白质交互调控染色质动力学和基因表达。染色质相关 RNA 免疫沉淀 (CARIP) 其次是下一代测序 (CARIP 序列) 是一种新的方法来调查与染色质蛋白相关的 rna, 而染色质免疫沉淀其次是下一代测序 (芯片序列) 是一种功能强大的基因组学技术, 用于在 ES 细胞的全球尺度上绘制出蛋白、转录因子和表观遗传修饰符的平移后修饰位置。在这里, 我们描述了执行 CARIP 和芯片序列的方法, 包括用于下一代测序的库构造, 以生成 ES 细胞中的全球染色质相关 RNA 和 epigenomic 图。

我们通过这个芯片序列协议6成功地审问了 ES 细胞中 H3K4me3、H3K4me2 和 KDM5B 的全基因组绑定。ES 细胞是在无馈线条件下培养的 (图 1), 并如上文所述交叉链接。超声波随后按照芯片序列协议步骤3.2 所述执行, 并通过在2% 琼脂糖凝胶上运行 DNA 进行评估 (图 2)。接下来, 按照步骤4中的描述执行芯片, 并按照步骤6中的?...

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

在这里, 我们描述了执行芯片序列和 CARIP 的方法, 包括用于下一代测序的库准备, 以生成 ES 细胞中的全球 epigenomic 和染色质相关的 RNA 图谱。

CARIP 和芯片序列: 鉴定胚胎干细胞中染色质相关 rna 和蛋白质-DNA 相互作用的方法

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

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

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

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

染色质监管机构和各国的全基因组快照<em&gt;爪</em&gt;通过胚胎的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 ...

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

小鼠胚胎干细胞的无血清单层培养神经细胞分化

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

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

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

心脏分化的表观遗传调控胚胎干细胞和组织

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

小鼠胚胎Hemogenic内皮细胞的分离

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

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

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

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

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

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

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

活完好斑马鱼的单细胞 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 ...