Name: chromatin immunoprecipitation (chip) using drosophila tissue
Uploaded: 2012-03-23T13:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP using Drosophila tissue at JoVE.com

笔者想感谢他们副科级赵博士的实验室研究院（NIH / NHLBI）的测序结果提供帮助。我们也想感谢UCSC基因组计划，基因组浏览器使用的可视化映射测序读取。 一直支持这项工作由R00HD055052 NIH的通路独立奖和从NICHD的，露西尔Parkard基金会，约翰·霍普金斯大学的启动资金到XC R01HD065816

（整个芯片过程大约需要两天。高通量测序芯片库的制备另需2-3天。） 1。解剖，并准备组织芯片实验（约1亿个细胞） <ol><li>冷PBS + 1蛋白酶抑制剂（溶解在1.5 mL的1X PBS 1蛋白酶抑制剂的鸡尾酒颗粒获得7X原液）在解剖组织的利益（例如果蝇精巢200双）+ PMSF（终浓度为100微克/毫升）。漂洗两次组织和悬浮在200μL与抑制剂的解决方案相同的PBS组织。 </li><li>?...

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	<li>Kharchenko, P. V., Alekseyenko, A. A., Schwartz, Y. B., Minoda, A., Riddle, N. C., Ernst, J., Sabo, P. J., Larschan, E., Gorchakov, A. A., Gu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analysis+of+the+chromatin+landscape+in+Drosophila+melanogaster.">Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.</a> Nature. 471, 480-485 (2011).</li><li>Filion, G. J., van Bemmel, J. G., Braunschweig, U., Talhout, W., Kind, J., Ward, L. D., Brugman, W., de Castro, I. J., Kerkhoven, R. M., Bussemaker, H. J., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+protein+location+mapping+reveals+five+principal+chromatin+types+in+Drosophila+cells.">Systematic protein location mapping reveals five principal chromatin types in Drosophila cells.</a> Cell. 143, 212-224 (2010).</li><li>Barski, A., Cuddapah, S., Cui, K., Roh, T. Y., Schones, D. E., Wang, Z., Wei, G., Chepelev, I., 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=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129, 823-837 (2007).</li><li>Chen, X., Lu, C., Prado, J. R., Eun, S. H., Fuller, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+changes+at+differentiation+gene+promoters+as+they+become+active+in+a+stem+cell+lineage.">Sequential changes at differentiation gene promoters as they become active in a stem cell lineage.</a> Development. 138, 2441-2450 (2011).</li><li>Gonczy, P., Matunis, E., DiNardo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bag-of-marbles+and+benign+gonial+cell+neoplasm+act+in+the+germline+to+restrict+proliferation+during+Drosophila+spermatogenesis.">bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis.</a> Development. 124, 4361-4371 (1997).</li><li>McKearin, D. M., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bag-of-marbles:+a+Drosophila+gene+required+to+initiate+both+male+and+female+gametogenesis.">bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis.</a> Genes Dev. 4, 2242-2251 (1990).</li><li>Gan, Q., Schones, D. E., Eun, S. H., Wei, G., Cui, K., Zhao, K., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monovalent+and+unpoised+status+of+most+genes+in+undifferentiated+cell-enriched+Drosophila+testis.">Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis.</a> Genome Biol. 11, 42-42 (2010).</li><li>Gan, Q., Chepelev, I., Wei, G., Tarayrah, L., Cui, K., Zhao, K., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+alternative+splicing+and+chromatin+structure+in+Drosophila+gonads+revealed+by+RNA-seq.">Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq.</a> Cell Res. 7, 763-783 (2010).</li></ol>

本议定书中所讨论的芯片分析的通用性，可用于在不同的组织，它提供了一个机会，在生物相关系统研究染色质状态。芯片实验中使用的细胞培养系统是方便执行，因为可以很容易地获得大量的细胞。然而，培养的细胞并不一定反映在多细胞环境中的细胞。通过使用活的动物解剖组织开发这一技术，我们可以解决许多问题，培养的细胞不能。 尽管本协议的有效性，有几个注?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>完成迷你蛋白酶抑制剂的鸡尾酒</td><td>罗氏公司</td><td> 11836153001 </td><td></td></tr><tr><td>甲醛（37％） </td><td> Supelco公司</td><td> 47083 - U </td><td></td></tr><tr><td> PMSF </td><td>西格玛</td><td> 78830 </td><td></td></tr><tr><td> kontes颗粒杵</td><td>菲舍尔科学</td><td> K749521-1590 </td><td></td></tr><tr><td> PCR纯化试剂盒</td><td> Qiagen公司</td><td> 28104 </td><td></td></tr><tr><td>线性聚丙烯酰胺</td><td>西格玛</td><td> 56575-1ML </td><td></td></tr><tr><td>糖原</td><td> Qiagen公司</td><td> 158930 </td><td></td></tr><tr><td> SYBR绿色/红霉素定量PCR预混</td><td> Fermentas公司</td><td> K0223 </td><td></td></tr><tr><td>迷你板微调</td><td> labnet </td><td> Z723533 </td><td></td></tr><tr><td>实时定量PCR系统</td><td>应用生物系统</td><td> 4351101 </td><td></td></tr><tr><td>体积小，超声波处理器</td><td> misonix </td><td> HS-XL2000 </td><td>型号停产</td></tr><tr><td>磁珠，蛋白A </td><td> Invitrogen公司</td><td> 100-01D </td><td></td></tr><tr><td> Dynamag磁铁</td><td> Invitrogen公司</td><td> 123-21D </td><td></td></tr><tr><td>酚：氯仿：生长素</td><td> Invitrogen公司</td><td> 15593-049 </td><td></td></tr><tr><td>震中的DNA完修理包</td><td>震中生物技术</td><td> ER0720 </td><td></td></tr><tr><td> MinElute Reactio列印清理套件</td><td> Qiagen公司</td><td> 28204 </td><td></td></tr><tr><td> Klenow片段（3&#39;→5&#39;外切） </td><td> New England Biolabs公司</td><td> M0212S </td><td></td></tr><tr><td> T4 DNA连接酶</td><td> Promega公司</td><td> M1794 </td><td></td></tr><tr><td>适配器寡核苷酸</td><td> Illumina公司</td><td> PE-400-1001 </td><td></td></tr><tr><td>配对末端引物1.0和2.0 </td><td> Illumina公司</td><td> 1001783 1001 784 </td><td></td></tr><tr><td>电子凝胶Electorphoresis系统</td><td> Invitrogen公司</td><td> G6512ST </td><td></td></tr><tr><td> 2X Phusion高频Mastermix </td><td> finnzymes </td><td>架F-531 </td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) using drosophila tissue

表观遗传学仍然是一个发展迅速的领域，如何染色质状态差，在不同类型的细胞在不同发育阶段的基因表达的研究。表观遗传调控，有利于广泛的生物过程，包括细胞分化的胚胎发育过程中和在成年后的动态平衡。在表观遗传学研究的一个重要战略，是研究各种组蛋白修饰和染色质的因素，如何调节基因的表达。为了解决这个问题，染色质免疫沉淀（ChIP）被广泛使用，以获得与细胞DNA的特定因素的关联的快照。 芯片技术作为起始原料，可在获得丰度和均匀性产生重复性的数据，通常使用的细胞培养。然而，有几个注意事项：首先，在培养皿中生长的细胞环境是从体内不同，因此可能在一个活的有机体，不能反映内源性细胞的染色质状态。其次，并非所有类型的细胞可以体外培养。有只有一个细胞系的数量有限，从中人们可以得到足够的材料芯片检测。 在这里，我们描述的方法做芯片实验用果蝇组织。起始原料是从一个活生生的动物解剖组织，从而能准确反映内源性染色质状态。这种方法有许多不同类型的组织的适应性将允许研究人员解决了很多生物体内 1，2的表观遗传调控的有关问题。此方法结合高通量测序技术（ChIP-SEQ），将进一步使研究人员能够获得表观景观。

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP using Drosophila tissue at JoVE.com

Chromatin Immunoprecipitation ChIP using Drosophila tissue

最近高通量测序技术，大大提高了染色质免疫沉淀（ChIP）实验的灵敏度，并促使其使用纯化的细胞或解剖组织的申请。在这里，我们划定使用芯片技术与方法<em&gt;果蝇</em&gt;组织，可以解决在一个特点的生物系统的内源性染色质状态。

染色质免疫沉淀（ChIP）使用果蝇组织

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Research

JoVE Journal

Biology

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

图案的使用生物倒装芯片的胚胎干细胞

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

科研

生物学

A Chromatin Assay for Human Brain Tissue

一个人脑组织中的染色质含量

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

从人类胚胎干细胞的染色质免疫沉淀

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

Methylated DNA Immunoprecipitation

甲基化的DNA免疫沉淀

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

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

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

生物质的使用限制金额高效的染色质免疫沉淀

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

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

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

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

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

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...