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>要解决的样品，加入5.5μL37％甲醛（在37℃的水浴在使用前1分钟的热烈甲醛）。在37°C孵育15分钟，涡之间，每隔5分钟。 </li><li>置于冰上2分钟解决的组织样本。冲洗450μLPBS（含蛋白酶抑制剂和PMSF）的2倍。样品现在可以是商店d在-20°C。重复清扫多次获得芯片足够的材料。 </li></ol> 2。准备与蛋白质-DNA共轭上清芯片检测<ol><li>从1.75 mL的Eppendorf管中的1.3步结合足够的样本。 </li><li>从组织样本PBS。加入裂解液（200微升的50 mM Tris-盐酸，pH值7.6，1毫米氯化钙，0.2％的Triton X-100或NP-40，5毫米丁酸，1X蛋白酶抑制剂的鸡尾酒）。添加新鲜PMSF裂解液（PMSF终浓度为0.5毫米）的股票解决方案。蓝色均质（费舍尔猫＃749521-1590）均质，直到组织没有任何聚集在室温（室温）孵育10分钟，完全游离于。 </li><li>超声：使用微尖sonicator功率20（Misonix HS-XL200）。在冰上超声为10秒，休息50秒。重复4-5次（应凭经验确定最佳超声时间，超声不再会产生较小的碎片对于华宏反对使用抗体的ChIP。ê修改，我们通常约200-300 bp的超声染色质转录因子或染色质调节（如Polycomb蛋白），我们通常超声200-1000 bp的染色质。然而，最佳片段大小应测试经验。注意：始终保持在ICE，甚至在超声分散，以防止样品加热管！ </li><li>加入1.8 mL的RIPA缓冲液（稀释样品10毫米的Tris-HCl，pH值7.6，1 mM的EDTA，0.1％SDS，0.1％钠 - 脱氧胆酸，1％的Triton X-100的蛋白酶抑制剂和PMSF，在相同浓度的描述先前）。保存作为输入控制40μL，加入2微升5M NaCl和孵育65°C过夜器（O / N）的逆转交联。 </li><li>共轭抗体珠，加入40μL蛋白A珠1.5毫升的Eppendorf管中，再加入600μL的PBS和岩石在4°C 2分钟，适用于磁铁珠，上清。加入100μL的PBS和利益的抗体（抗体量应确定empirically）。在室温下孵育1小时或4°C间为4小时。 </li><li>用磁铁珠上清，加入1ml提取物的染色质从2.4步的珠子。旋转4°CO / N </li><li>芯片样品申请磁铁和上清。珠洗，每次10分钟，在以下4个缓冲区°C间： RIPA缓冲液1毫升的2倍[1.89毫升的RIPA缓冲液+ 315μL7X蛋白酶抑制剂，20μLPMSF]; RIPA缓冲液1毫升的2倍与±0.3 M氯化钠1.89毫升RIPA缓冲液220μL，3 M氯化钠; 用1毫升的LiCl缓冲区（0.25M氯化锂，0.5％NP40，0.5％NaDOC），2倍; 1X与1毫升的1X TE + 0.2％的Triton X-100; 1X与1毫升的1X TE。 </li><li>为了扭转交联，珠悬浮在100μLTE缓冲+ 3μL的10％SDS + 5μL蛋白酶K（20毫克/毫升）。在65孵育°CO / N </li><li> *珠子磁铁申请上清转移到一个新的管。上清包含的DNA样本。洗净与100μL的TE + 0.5米珠NaCl和结合两个上清。 </li><li>酚 - 氯仿提取DNA样本。加入200μL的苯酚：氯仿：生长素（25:24:1）混合和涡。在室温下5分钟14K旋转。另外，使用Qiagen公司的PCR纯化试剂盒提取DNA可。 </li><li>转移到一个新的管上清/水层，也可用于线性丙烯酰胺加入到终浓度为20微克/毫升（或者1μL在20毫克/毫升，每1毫升上清糖原，糖原被用于随后的定量PCR或PCR分析，而线性丙烯酰胺用于测序分析。）加入20μL的3M醋酸钠和500μL100％的乙醇。拌匀，孵育80°Ç10分钟。在最大速度旋转，在4°C. 20分钟</li><li>除去上清液，用300μL，70％的乙醇。空气干燥，重悬在50μLTE缓冲液样本。样品现在可以保存在-20°C。样品可用于定量PCR（qPCR）检测（ 图1）。 </li></ol> 3A。使用定量PCR分析DNA芯片-ED <ol><li>基因组DNA在以下浓度稀释，作出标准曲线：未稀释，1/10，1/100，1/1000，1/5000。 </li><li>对于每对引物，建立一式两份，一个步骤3A.1，输入控制芯片DNA基因组DNA 96孔板的qPCR： 10μL的2倍SYBR PCR混合物（Fermentas公司，K0222）; 1μL，10微米正向引物; 1μL，10μM的反向引物; xμL的DNA芯片（输入控制，或基因组DNA）; ŸμL无核酸酶H 2 O调整至20μL的反应体积。 </li><li>降速迷你板微调（Labnet）96孔板。 </li><li>执行的qPCR的ABI 7300实时PCR系统使用以下条件（或其他实时PCR系统）： 第1阶段：50°C，2分，1个周期 第2阶段：95°C为10分钟，1个周期; 第三阶段：95℃1分钟15秒，60°，40°Cycles; 第四阶段（分解阶段）：95℃15秒，60°C下1分钟，95°15秒ç。 </li><li>检查解离曲线：在热分解图的峰值，表明了从单一PCR反应扩增。多个峰值，表明非特定产品的引物，不应使用qPCR数据。 </li><li>确定每个引物的PCR反应的指数放大的线性相位，计算公式，解决DNA含量，根据CT（循环阈值）值，这是基于使用稀释的基因组DNA在3A系列标准曲线。 1。 </li><li>如果Ct值的线性范围内的DNA芯片-ED和输入控制CT值，计算DNA芯片-ED与输入的富集，根据DNA芯片-ED和输入控制在2.4稀释因素。 </li></ol> 3B。放大为高通量测序的DNA芯片-ED。 <ol><li>年底修复的DNA组合芯片-ED（使用震中的DNA完修理包），冰上成立： 1-34μL的基因组DNA的步骤2.12（0.1至5微克） 5μL10倍完修复缓冲区 5μL2.5毫米的dNTP混合 5μL10 mM的ATP x微升H 2 O调整反应体积49μL 1μL最终修复酶组合（T4 DNA聚合酶+ T4聚核苷酸激酶） 在室温45分钟孵育。净化使用Qiagen公司MinElute反应的反应 清理套件。在30μL洗脱缓冲液（EB），洗脱。 </li><li> 3&#39;末端添加一个-出挑： 30μL洗脱DNA的产品从步骤3b.1 5μL10X的NEB缓冲＃2 10微升1毫米的dATP 2μLDH 2 O的 3μL5 U /μLKlenow片段（3&#39;→5&#39;外切） 在37°C孵育30分钟净化反应使用MinElute ReactionCleanup套件。洗脱在10μL的EB。 </li><li>公司Solexa链接结扎： 10μL洗脱DNA的步骤3B.2 10μLDDH 2 O的 2.5＆亩;升10倍T4 DNA连接酶缓冲 1μL适配器从Illumina公司（从股票1/10-diluted的寡核苷酸组合） 2.5μLT4 DNA连接酶（400单位/μL） 1小时在室温下孵育。净化反应使用MinElute ReactionCleanup套件。洗脱在20μL的EB。样品现在可以保存在-20°C。 </li><li>运行使用的E-凝胶仪器的步骤3b.3洗脱的DNA样本。凝胶在300-500 bp的条带隔离和净化使用Qiagen公司的胶回收试剂盒（这一步消除〜125 bp的自我结扎连接器，适配器寡结扎）。在12μL的EB洗脱。 </li><li>放大库使用配对末端从Illumina公司（PE）引物： 10.5微升洗脱DNA的步骤3b.4 12.5μL的master mix（2X Phusion HF，Finnzymes） 1 PE1的PCR引物1μL 1 PE2的PCR引物2μL PCR条件： ℃30 98°秒 （98℃10秒，65℃30秒，72°℃30秒），重复20个循环 72℃5分钟。 </li><li>步骤3b.5样品可以用于芯片SEQ后，选择适当大小的DNA（300-500 bp）的使用标准的2％琼脂糖凝胶。样品现在可以保存在-20°C。最好是在一个单一的凝胶，以防止污染隔离每个芯片样品。可以提交样品的高通量测序。 </li></ol> 3C。公司Solexa管道分析<ol><li> Illumina的Genome Analyzer的管道（GA）的25 bp的测序读得到。 </li><li>所有读取数据对齐的果蝇基因组（DM3）使用伊兰（高效核苷酸数据的地方对准）软件，允许最多两个与参考序列的不匹配。 </li><li>唯一映射读取被保留用于下游分析。多个相同的读取，最多三份被保留，以减少PCR扩增偏见的可能性。 </li><li>联大管道输出转换为浏览器扩展的数据文件（床）。 </li><li>要生成假发文件上传到加州大学圣克鲁兹分校的可视化浏览器，我们用一个python的以股代息已在以前出版3 4基点，作为窗口的大小和160 bp的DNA片段的大小。 </li><li>分为沉默和表达的基因的果蝇基因中，我们使用数字量称为RPKM（读取/每碱基合并外显子区域/每百万映射读取）。沉默与RPKM≥1被归类到表达团体，列为低，中，高表达水平的基因可分为三个亚组和基因的基因被列为与RPKM = 0，每个组有大约相同数量的基因。 </li><li>要比较的组蛋白修饰水平和基因表达，我们用一个Python脚本，已在以前的出版物3。 </li></ol> 4。代表结果芯片-qPCR的结果，使用BAM的例子（弹子袋）突变<html>睾丸显示在图1 4。在BAM睾丸，在过渡增殖的精原细胞分化精5，6是一个失败。我们使用BAM睾丸未分化的生殖细胞，这是这个组织类型丰富的来源。精子分化所需的分化的基因，如男性特有的转录因子87（mst87F），唐璜（DJ），模糊洋葱（fzo），是不是表示在BAM睾丸。这些基因是高度浓缩的镇压H3K27me3组蛋白修饰（ 图1A），但缺乏积极H3K4me3的组蛋白修饰（ 图1B），染色质的签名，我们称为“单价”7。 H3K27me3或H3K4me3的任何富集确定一个组成细胞周期蛋白A（CycA）基因表达的正常化。 内容“&gt;的ChIP-seq的分析使用BAM突变睾丸（ 图2）验证qPCR的结果如图1所示。对于三个测试终端分化的基因，DJ和fzo的mst87F的抗体相同的一组（即镇压H3K27me3和H3K4me3的积极）高度富集，他们的基因组区域与H3K27me3但不是H3K4me3的（ 图2A-2C）。相比之下，组成表示CycA基因的H3K4me3的具有显着的，但很少H3K27me3其转录起始位点（TSS），（ 图2D）附近的约束力。 此外，四个班的差异表达基因的转录起始位置跟附近的H3K27me3和H3K4me3的芯片配置文件是每个组蛋白修饰与功能一致。 图3A所示，H3K27me3下游的起始位置跟富集与基因表达水平呈负相关。沉默的基因，而最高的H3K27me3高表达的基因有没有H3K27me3约束力。这些数据对基因表达的H3K27me3压制作用是一致的。相比之下，周围的起始位置跟H3K4me3的富集表明，与对面相关基因的表达水平（ 图3B），对基因表达的H3K4me3的积极作用。 <img alt="图1" src="/files/ftp_upload/3745/3745fig1.jpg" /> 图1。qPCR分析，对镇压H3K27me3组蛋白修饰（A）或积极H3K4me3的组蛋白修饰（B）在未分化的细胞富集BAM突变睾丸抗体的DNA芯片-ED。 （一）在BAM睾丸分化的基因（Mst87F DJ，fzo）的富集与H3K27me3镇压组蛋白修饰。 （二）分化的基因被耗尽H3K4me3的积极标志。第一级别的靶基因（Mst87F，DJ或fzo的 ）DNA芯片（芯片的DNA /输入）正常化的利<html> EL的在芯片控制CycA基因的DNA。误差棒表示标准偏差由三个独立的生物复制。 <img alt="图2" src="/files/ftp_upload/3745/3745fig2.jpg" /> 图2。 UCSC基因组浏览器H3K27me3快照和H3K4me3的跨越（一）Mst87F（二）DJ和（三），（四）fzo CycA基因。（d）中盘旋H3K27me3富集地区的整个基因组区域的浓缩反映了染色质状态重叠基因CG7264，这是弱旅巴姆表示睾丸（RPKM = 1），但高表达野生型的睾丸（RPKM = 130）8（SEQ芯片数据从7）。定量PCR图1芯片结果的分析中使用的探针标记在每个小区的底部。 <a href="http://www.jove.com/files/ftp_upload/3745/3745fig2large.jpg" target="_blank">点击这里查看大图</a> 。 tp_upload/3745/3745fig3.jpg“/&gt; 图3。芯片SEQ配置使用BAM H3K27me3及H3K4me3的睾丸 7。四个基因组（7509个基因）进行分类，根据他们的基因表达水平，基于RNA-seq的结果8。绘制基因的代表类的特定组蛋白修饰的富集，从3KB上游3KB下游的转录起始位点（起始位置跟）使用序列。这会产生一个丰富 ​​的（一）的H3K27me3（K27次）和（乙）H3K4me3的（K4），各组SEQ芯片分析。的个人资料<a href="http://www.jove.com/files/ftp_upload/3745/3745fig3large.jpg" target="_blank">点击这里查看大图</a> 。

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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 ...

chromatin-immunoprecipitation-chip-using-drosophila-tissue

Research

JoVE Journal

Biology

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

25.0K 次观看.  Johns Hopkins University. 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果蝇组织，可以解决在一个特点的生物系统的内源性染色质状态。

视频: Chromatin Immunoprecipitation ChIP using Drosophila tissue

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

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

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

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

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

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

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

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

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

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

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

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

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

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

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

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

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

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

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

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

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

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)

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

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

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

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

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

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 ...