Name: genome-wide analysis of histone modifications distribution using the chromatin immunoprecipitation sequencing method in magnaporthe oryzae
Uploaded: 2021-06-02T14:00:00
Description: Watch this Scientific Journal Video about Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae at JoVE.com

这项工作得到了中国国家自然科学基金（31871638号赠款）、北京农业大学（YQ201603）、北京市教委科学项目（KM201610020005）、BUA高水平科研培育项目（GJB2021005）的支持。

1. 从奥里扎伊预制原体
<ol><li>准备燕麦片番茄（OTA）。<ol>
<li>重30-50克燕麦片，在800mL水中煮沸（ddH2O），煮20分钟。过滤通过两层纱布，并采取过滤。</li>
		<li>挑选成熟的西红柿并剥皮。挤压果汁，过滤两层纱布，收集150ml的过滤果汁。</li>
		<li>将番茄汁和准备好的燕麦过滤液彻底混合，加入 ddH2O 至 1000 mL。</li>
		<li>在 500 mL 圆锥形烧瓶中加入 250 mL 的 OTA 和 2.5 克的醋粉，?...

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+modifications+of+histones+during+development+and+disease+pathogenesis.">Covalent modifications of histones during development and disease pathogenesis.</a> Nature Structural and Molecular Biology. 14 (11), 1008-1016 (2007).</li><li>Shilatifard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+implementation+and+physiological+roles+for+histone+H3+lysine+4+(H3K4)+methylation.">Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation.</a> Current Opinion in Cell Biology. 20 (3), 341-348 (2008).</li><li>Berger, 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=The+complex+language+of+chromatin+regulation+during+transcription.">The complex language of chromatin regulation during transcription.</a> Nature. 447 (7143), 407-412 (2007).</li><li>Bernstein, B. E., 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+mammalian+epigenome.">The mammalian epigenome.</a> Cell. 128 (4), 669-681 (2007).</li><li>Weake, V. M., Workman, 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=Histone+ubiquitination:+triggering+gene+activity.">Histone ubiquitination: triggering gene activity.</a> Molecular Cell. 29 (6), 653-663 (2008).</li><li>Workman, J. L., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+nucleosome+structure+as+a+mechanism+of+transcriptional+regulation.">Alteration of nucleosome structure as a mechanism of transcriptional regulation.</a> Annual Review of Biochemistry. 67 (1), 545-579 (2003).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128 (4), 693-705 (2007).</li><li>Akhtar, J., More, P., Albrecht, S. <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+from+limited+starting+material+of+K562+cells+and+Drosophila+neuroblasts+using+tagmentation+assisted+fragmentation+approach.">ChIP-Seq from limited starting material of K562 cells and Drosophila neuroblasts using tagmentation assisted fragmentation approach.</a> Bio-protocol. 10 (4), 3520 (2020).</li><li>Steinhauser, S., Kurzawa, N., Eils, R., Herrmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+comparison+of+tools+of+differential+ChIP-seq+analysis.">A comprehensive comparison of tools of differential ChIP-seq analysis.</a> Briefings in Bioinformatics. 17 (6), 953-966 (2016).</li><li>Kieu, T., 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=MoSET1+(histone+H3K4+methyltransferase+in+Magnaporthe+oryzae)+regulates+global+gene+expression+during+infection-related+morphogenesis.">MoSET1 (histone H3K4 methyltransferase in Magnaporthe oryzae) regulates global gene expression during infection-related morphogenesis.</a> Plos Genetics. 11 (7), 11005385 (2015).</li><li>Vu, B. V., Pham, K. T., Nakayashiki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-induced+transcriptional+activation+of+the+MoCel7C+cellulase+gene+is+associated+with+methylation+of+histone+H3+at+lysine+4+in+the+rice+blast+fungus+Magnaporthe+oryzae.">Substrate-induced transcriptional activation of the MoCel7C cellulase gene is associated with methylation of histone H3 at lysine 4 in the rice blast fungus Magnaporthe oryzae.</a> Applied and Environmental Microbiology. 79 (21), 6823-6832 (2013).</li><li>Kazan, K., Gardiner, D. M., Manners, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+trail+of+a+cereal+killer:+recent+advances+in+Fusarium+graminearum+pathogenomics+and+host+resistance.">On the trail of a cereal killer: recent advances in Fusarium graminearum pathogenomics and host resistance.</a> Molecular Plant Pathology. 13 (4), 399-413 (2012).</li><li>Wang, G. H., 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+AMT1+arginine+methyltransferase+gene+is+important+for+plant+infection+and+normal+hyphal+growth+in+Fusarium+graminearum.">The AMT1 arginine methyltransferase gene is important for plant infection and normal hyphal growth in Fusarium graminearum.</a> PLoS One. 7 (5), 38324 (2012).</li><li>Liu, 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=Histone+H3K4+methylation+regulates+hyphal+growth,+secondary+metabolism+and+multiple+stress+responses+in+Fusarium+graminearum.">Histone H3K4 methylation regulates hyphal growth, secondary metabolism and multiple stress responses in Fusarium graminearum.</a> Environmental Microbiology. 17 (11), 4615-4630 (2016).</li><li>Zhang, M. 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=The+plant+infection+test:+spray+and+wound-mediated+inoculation+with+the+plant+pathogen+Magnaporthe+grisea.">The plant infection test: spray and wound-mediated inoculation with the plant pathogen Magnaporthe grisea.</a> Journal of Visualized Experiments. (138), e57675 (2018).</li><li>Connolly, L. R., Smith, K. M., Freitag, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fusarium+graminearum+histone+H3K27+methyltransferase+KMT6+regulates+development+and+expression+of+secondary+metabolite+gene+clusters.">The Fusarium graminearum histone H3K27 methyltransferase KMT6 regulates development and expression of secondary metabolite gene clusters.</a> PloS Genetics. 9 (10), 1003916 (2013).</li><li>Palmer, J. 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=Loss+of+CclA,+required+for+histone+3+lysine+4+methylation,+decreases+growth+but+increases+secondary+metabolite+production+in+Aspergillus+fumigatus.">Loss of CclA, required for histone 3 lysine 4 methylation, decreases growth but increases secondary metabolite production in Aspergillus fumigatus.</a> PeerJ. 1, 4 (2013).</li><li>Ding, S. 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+Tig1+histone+deacetylase+complex+regulates+infectious+growth+in+the+rice+blast+fungus+Magnaporthe+oryzae.">The Tig1 histone deacetylase complex regulates infectious growth in the rice blast fungus Magnaporthe oryzae.</a> The Plant Cell. 22 (7), 2495-2508 (2010).</li><li>Dean, R. 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=The+genome+sequence+of+the+rice+blast+fungus+Magnaporthe+grisea.">The genome sequence of the rice blast fungus Magnaporthe grisea.</a> Nature. 434 (7036), 980-986 (2005).</li><li>Allis, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+nomenclature+for+chromatin-modifying+enzymes.">New nomenclature for chromatin-modifying enzymes.</a> Cell. 131 (4), 633-636 (2007).</li><li>Shilatifard, 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+COMPASS+family+of+histone+H3K4+methylases:+mechanisms+of+regulation+in+development+and+disease+pathogenesis.">The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis.</a> Annual Review of Biochemistry. 81, 65-95 (2012).</li><li>Zhou, S. 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最近，ChIP-seq已成为一种广泛使用的基因组分析方法，用于确定由特定体调修改的TF或浓缩站点的结合部位。与以往的 ChIP-seq 技术相比，新的 ChIP-seq 技术具有高度的灵敏性和灵活性。结果以高分辨率提供，不产生负面影响，例如核酸非特异性杂交引起的噪声信号。虽然这是一种常见的基因表达分析，但许多计算方法已经得到验证，ChIP-seq 数据在噪声和变异性方面的复杂性使得 ChIP-seq 尤其难以克服...

表观遗传学是基因研究的一个分支，它是指基因表达的遗传变化，而不改变基因的核苷酸序列。越来越多的研究表明，表观遗传调节在真核细胞的生长发育中起着重要作用，包括通过折叠和组装成1、2等高阶结构的动态过程调节和影响基因表达的染色质。色度素重塑和共价色蛋白修饰通过色度素聚合物的变异影响和调节染色质的功能和结构，从而达到调节基因表达3、4、5、6的功能。平石的后翻译修改包括乙酰化、磷酸化、甲基化、单体化、相扑和ADP核糖化7、8、9。Histone H3K4 甲基化，特别是三甲基化，已映射到转录启动站点，它与真核生物10、11中的转录复制、重组、修复和 RNA 处理相关联。
ChIP-seq技术于2007年引进，现已成为全基因组转录调节和表观遗传机制12、13分析的实验标准。这种方法适用于全基因组尺度，并用于获取组蛋白或转录因子相互作用信息，包括DNA结合蛋白的DNA片段。任何与感兴趣的蛋白质相交的DNA序列都会作为染色质复合物的一部分沉淀。新一代测序技术也用于测序36-100桶DNA，然后与相应的目标基因组匹配。
在植物病原真菌中，研究最近开始研究希司他甲基化修饰如何在致病过程中调节其目标基因。先前的一些研究证明，与平方甲酶相关的基因的调控主要反映在基因沉默和催化二次代谢物（SM）的产生上。MoSet1 是M. oryzae中的 H3K4 甲基酶。该基因的敲除导致H3K4me3修改14的完全删除。与野生型菌株相比，突变体中基因MoCEL7C的表达在CMC诱导状态和非诱导状态（葡萄糖或大提琴）中受到抑制，MoCEL7C的表达增加15。在富萨里姆胶质中，KMT6可以促进H3K27me3的甲基化改造，调节真菌的正常发育，并有助于调节含有SM基因簇16、17、18、19的"神秘基因组"。2013年，康诺利报告说，H3K9和H3K27甲基化通过二次代谢物和调节目标基因抑制的效应因子调节真菌的致病过程。在阿斯珀吉卢斯，H3K4me2和H3K4me3组的改变与基因活化有关，在控制SM基因簇21的染色质水平调节方面起着重要作用。在M.oryzae，Tig1（酵母和哺乳动物中的Tig1同源）是一个哈德（石质去乙酰酶）22。Tig1基因的敲除导致空突变体中致病性和孢子生产能力的完全丧失。它对过氧环境更敏感，不能产生感染性催眠22。
由M.oryzae引起的稻米爆炸是世界上大多数水稻种植区最严重的水稻疾病之一。由于其代表性的感染过程，M.oryzae与许多重要致病真菌的感染过程相似。由于真菌可以轻易地进行分子遗传操作，因此已成为研究真菌与植物相互作用的模型生物体。阻断M. oryzae感染过程的每一步都可能导致感染不成功。感染过程中的形态变化受整个基因组功能和基因转录的严格控制。其中，表观遗传修饰，如平石甲基化，在功能基因的转录调节中起着至关重要的作用。然而，到目前为止，在M.oryzae发病基因转录中，对表观遗传修饰的分子机制的研究很少，如平石甲基化和平石乙酰化。因此，在研究这些致病相关基因的上下游调控网络的同时，进一步完善水稻爆菌的表观遗传调控机制，将有助于制定水稻爆破防治策略。
随着ChIP-seq等功能基因组学的发展，特别是在表观遗传学方面，这种高通量数据采集方法加速了染色体的研究。利用ChIP-seq实验技术，可以识别 出M.oryzae 和其他丝状真菌的全基因组分布（如H3K4me3、H3K27me3、H3K9me3）。因此，这种方法可以帮助阐明表观遗传修饰在植物病理学中的真菌发病机制中如何调节其候选靶基因的分子机制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>acidic casein hydrolysate</td>
			<td>WAKO</td>
			<td>65072-00-6</td>
			<td>Medium configuration</td>
		</tr>
		<tr>
			<td>agar powder</td>
			<td>scientan</td>
			<td>9002-18-0</td>
			<td>Medium configuration</td>
		</tr>
		<tr>
			<td>deoxycholic acid</td>
			<td>MedChemExpress</td>
			<td>83-44-3</td>
			<td>protein and dissolution</td>
		</tr>
		<tr>
			<td>DNA End-Repair kit</td>
			<td>NovoBiotec</td>
			<td>ER81050</td>
			<td>Repair DNA or cDNA damaged by enzymatic or mechanical shearing</td>
		</tr>
		<tr>
			<td>Dynabeads</td>
			<td>Invitrogen</td>
			<td>no.100.02D</td>
			<td>Binding target</td>
		</tr>
		<tr>
			<td>EB buffer</td>
			<td>JIMEI</td>
			<td>JC2174</td>
			<td>Membrane and liquid</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher</td>
			<td>AM9912</td>
			<td>protease inhibitor</td>
		</tr>
		<tr>
			<td>enzymatic casein hydrolysate</td>
			<td>Sigma</td>
			<td>91079-40-2</td>
			<td>Medium configuration</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Sigma</td>
			<td>50-99-7</td>
			<td>Medium configuration</td>
		</tr>
		<tr>
			<td>glycogen</td>
			<td>ThermoFisher</td>
			<td>AM9510</td>
			<td>Precipitant action</td>
		</tr>
		<tr>
			<td>H3K4me3 antibodies</td>
			<td>Abcam</td>
			<td>ab8580</td>
			<td>Immune response to H3K4me3 protein</td>
		</tr>
		<tr>
			<td>illumina Genome Analyzer</td>
			<td>illumina</td>
			<td>illumina Hiseq 2000</td>
			<td>Large configuration</td>
		</tr>
		<tr>
			<td>Illumina PCR primers</td>
			<td>illumina</td>
			<td>CleanPlex</td>
			<td>Random universal primer</td>
		</tr>
		<tr>
			<td>isoamyl alcohol</td>
			<td>chemical book</td>
			<td>30899-19-5</td>
			<td>Purified DNA</td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>ThermoFisher</td>
			<td>AM9480</td>
			<td>specific removal RNA</td>
		</tr>
		<tr>
			<td>lysing enzymes</td>
			<td>Sigma</td>
			<td>L1412-10G</td>
			<td>cell lysis buffer</td>
		</tr>
		<tr>
			<td>Mouse IgG</td>
			<td>Yeasen</td>
			<td>36111ES10</td>
			<td>Animal normal immunoglobulin</td>
		</tr>
		<tr>
			<td>NaCl solution</td>
			<td>ThermoFisher</td>
			<td>7647-14-5</td>
			<td>Medium configuration</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Seebio</td>
			<td>SH30173.08*</td>
			<td>preparation of protein complex eluent</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>ThermoFisher</td>
			<td>85124</td>
			<td>cell lysate to promote cell lysis</td>
		</tr>
		<tr>
			<td>PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>The purification procedure removes primers from DNA samples</td>
		</tr>
		<tr>
			<td>protease inhibitors</td>
			<td>ThermoFisher</td>
			<td>A32965</td>
			<td>A protein inhibitor that decreases protein activity</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>ThermoFisher</td>
			<td>AM2546</td>
			<td>DNA Extraction Reagent</td>
		</tr>
		<tr>
			<td>Qubit 4.0</td>
			<td>ThermoFisher</td>
			<td>Q33226</td>
			<td>Medium configuration</td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher</td>
			<td>9806S</td>
			<td>cell lysis buffer</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>ThermoFisher</td>
			<td>AM2271</td>
			<td>Purified DNA</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>ThermoFisher</td>
			<td>AM9820</td>
			<td>cover up the charge differences</td>
		</tr>
		<tr>
			<td>sodium acetate solution</td>
			<td>ThermoFisher</td>
			<td>R1181</td>
			<td>Acetic acid buffer</td>
		</tr>
		<tr>
			<td>sodium deoxycholate</td>
			<td>ThermoFisher</td>
			<td>89904</td>
			<td>inhibition of protease degradation</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>ThermoFisher</td>
			<td>EL0011</td>
			<td>Under the condition of ATP as coenzyme, DNA ligase</td>
		</tr>
		<tr>
			<td>T4 DNA ligase buffer</td>
			<td>ThermoFisher</td>
			<td>B69</td>
			<td>DNA ligase buffer</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>ThermoFisher</td>
			<td>1185-53-1</td>
			<td>buffer action</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher</td>
			<td>HFH10</td>
			<td>keep the membrane protein stable</td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td>Medium configuration</td>
		</tr>
	</tbody>
</table>

genome-wide analysis of histone modifications distribution using the chromatin immunoprecipitation sequencing method in magnaporthe oryzae

色度素免疫沉淀测序 （ChIP-seq） 是一种强大且广泛使用的分子技术，用于绘制转录因子 （TFs）、染色质调节器和色石修饰的全基因组位置，以及检测整个基因组以发现 TF 结合模式和色石后翻译修饰。染色质修饰活动，如色蛋白甲基化，经常被招募到特定的基因调控序列中，导致染色质结构的局部变化，并导致特定的转录效应。稻米爆炸是全世界水稻上毁灭性的真菌病，是研究真菌与植物相互作用的示范系统。然而，在 麦格纳波特奥里扎伊 ，如何调节其毒性基因的分子机制仍然难以捉摸。更多的研究人员需要使用ChIP-seq来研究组蛋白表观遗传修饰如何调节他们的目标基因。ChIP-seq也广泛用于研究动物和植物中蛋白质与DNA之间的相互作用，但在植物病理学领域应用较少，尚未得到很好的开发。本文介绍了ChIP-seq的实验过程和操作方法，以确定与 M.oryzae 功能目标基因结合的希司他甲基化（如H3K4me3）的全基因组分布。在这里，我们提出了一个协议，以分析全基因组分布的色石修改，它可以识别新的目标基因在M.oryzae和其他丝状真菌的发病机制。

ChIP-seq 方法的整个流程图如图1所示。ChIP-seq实验使用抗体对H3K4me3在野生型菌株P131和三个空突变菌株，这是没有mobre2，mospp1和moswd2基因，以验证整个基因组的histone H3K4me3分布在M.oryzae的配置文件。 野生型菌株的原体，+mobre2，+mospp1和+moswd2，在25%W，输出3s，停止5s，在4°C准备和声波。 此外，染色质被免疫净化与H3K4me3抗体和发电机...

Watch this Scientific Journal Video about Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae at JoVE.com

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

在这里，我们提出了一个协议，以分析全基因组分布的色石修改，它可以识别新的目标基因在M.oryzae和其他丝状真菌的发病机制。

使用麦格纳波特奥里扎伊的色度免疫沉淀测序方法对希斯通修饰分布进行全基因组分析

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...

这种方法可以帮助阐明在植物病理学真菌发病过程中通过表观遗传修饰调节候选目标基因的分子机制。色度素免疫沉淀测序技术可以有效地检测与整个基因组中的组织素和转录因子相互作用的DNA片段。与其他方法相比，它可以提高效率和分辨率。首先，使用100微升移液器在燕麦番茄阿格尔板中加入100微升液体完全介质。使用接种循环，刮野生类型菌株和淘汰菌株的肌膜。收集肌裂碎片，并将其转移到250毫升的液体完全介质。用500毫升0.7摩尔氯化钠溶液清洗真菌水眠。然后收集真菌真菌，并称量它。每克真菌肌塞添加约一毫升裂解酶渗透溶液。将水眠放在28摄氏度下三到四个小时，在150 RPM处摇晃。用50毫升0.7摩尔氯化钠溶液清洗切片水眠。将样品离心，小心丢弃超高纳特。将前列座重新悬挂在 20 毫升 0.7 摩尔氯化钠溶液中。加入55微升37%甲醛滴到两毫升氯化钠缓冲器含有前列腺素的交叉连接，直到最终浓度为1%以淬火未反应的甲醛，在每根管子中加入20微升10倍甘油。离心后小心丢弃超高纳特。将颗粒重新悬挂在 0.7 摩尔氯化钠溶液的一毫升中。离心后小心丢弃超高纳特，并将颗粒重新悬挂在 RIPA 缓冲器的 750 微升中。加入37.5微升20倍蛋白酶抑制剂。使用超声波同质化器通过声波剪切染色素8分钟。样品被超声波分解后，取出部分样品作为输入，其中包含样品被声波化后释放的所有DNA和蛋白质。要分析DNA片段的长度，在声波化后运行1%的玫瑰凝胶电泳。离心后，将离心超离心剂转移到1.5毫升离心机管中，并储存在负80摄氏度下供以后使用。在进行 IP 实验之前，用 RIPA 缓冲器的一倍将每个染色质样本稀释为 1 比 10。派佩特50微升的超级准磁蛋白珠成两毫升离心机管。将管子放在磁架上，让磁珠沉淀。然后取出超那剂。在管子中加入一毫升预冷的 RIPA 缓冲器，并洗三次超级准磁蛋白珠。将管子放在磁架上，取出超高分子。在每个管子中加入 100 个 1 倍 RIPA 缓冲器的微升。在管中加入30微升染色素样品、100微升超准磁蛋白珠和4个H3K4me3抗体微升，混合均良好。将样品放在旋转摇床上，在摄氏4度、30倍G.的高温下在一夜之间孵育它们。 通过将珠子重新悬浮在一毫升的RIPA缓冲器中，洗净超级准磁蛋白珠抗体和染色质复合物。用一毫升低盐免疫复合洗涤缓冲器清洗样品，然后用一毫升高盐免疫复合洗涤缓冲器清洗样品。用一毫升氯化锂缓冲器冲洗珠子，取出超高分子。然后在管子中加入一毫升的 TE 缓冲器。再次用一毫升的TE缓冲器放置管子，用移液器取出超强的管子。在每个离心机管中加入100微升的弹性缓冲器。在65摄氏度下进行15分钟的放射。离心机在摄氏1万倍时在4摄氏度下离心一分钟，并将超高离心剂收集到新的离心机管中。在所有管子中加入8个5摩尔氯化钠的微升，在65摄氏度下孵育4至5小时或过夜，以扭转DNA蛋白的交叉联系。加入一微升Rnase A，在37摄氏度下孵育30分钟。在每个管子中加入四微升蛋白酶K，在45摄氏度下孵育一至两个小时。在离心机管中加入550微升的氯甲酸酯和同酰醇混合物。离心机 15 分钟， 10， 0 倍 G， 并吸气超纳特。在管中加入1/10卷三摩尔醋酸钠溶液、2.5卷绝对乙醇和三微升糖原。将样品放在负20摄氏度的冰箱中过夜，以便降水。第二天，离心管并丢弃超纳特。用一毫升新鲜制备的75%乙醇用1万倍G.添加50微升无菌去离子水，以充分溶解沉淀物，将颗粒清洗三次。修复DNA端，使用DNA端修复套件生成钝端DNA，并在文本手稿中提及的说明。要在三个主要端添加腺苷基，在 0.2 微升 PCR 离心机管中加入 30 微升 DNA、2 个水微升、5 个 10 倍 Taq 缓冲器、1 个 1 毫摩尔 dATP 微升和 3 个 Taq DNA 聚合酶微升。混合它们，在 72 摄氏度的 PCR 机器中反应 10 分钟。通过将 10 微升 DNA、9.9 微升水、2.5 微升 T4 DNA 韧带缓冲器、0.1 微升适配器寡头混合和 2.5 微升 T4 DNA 利加在 0.2 微升 PCR 离心机管中混合，执行链接器结结。在16摄氏度下孵育管子4小时。要使用 PCR 引数放大 DNA，添加 10.5 微升 DNA，12.5 微升的两倍高保真主组合。PCR 引引器 PE 1.0 的微升和 PCR 引漆 PE 2.0 到 0.2 微升 PCR 离心机管的微升，混合良好。mobre2、mospp1 和 moswd2 删除突变体的 H3K4me3 信号在其功能目标区域显著减少。与野生型菌株P131相比，富集H3K4me3染色质免疫沉淀测序的信号在mobre2、mospp1和moswd2中读取，删除突变体的信号大为减少。在此程序之后，可以执行芯片上的芯片和芯片 qPCR。它们通常用于筛选蛋白质的下游目标基因，并在已知的基因组结合点研究蛋白质DNA相互作用。

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Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Unit 1

Structure and Organization of DNA

SCIENTISTS IN ACTION

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

科研

遗传学

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

染色质免疫沉淀（ChIP）在小鼠T细胞系

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

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

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

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

低丰度胚胎标本的染色质沉淀 (芯片) 协议

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

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

ATAC 基因组在原发性人类 T 淋巴细胞中的染色体全易接近染色质

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

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

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

启动子捕获高分辨率, 全基因组的启动子交互分析

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

小鼠棕色脂肪组织的染色质免疫沉淀

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...