Name: generation of high quality chromatin immunoprecipitation dna template for high-throughput sequencing (chip-seq)
Uploaded: 2013-04-19T13:00:00
Description: Watch this Scientific Journal Video about Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq at JoVE.com

这项工作是支持学院发展奖由来自费城儿童医院。

1。跨链接染色的<ol><li> 100x20mm细胞培养皿中的细胞生长。细胞的量的范围可以从1到10百万个细胞每皿取决于细胞的类型。约2万个细胞就足够一个免疫沉淀。 </li><li>交叉链接的细胞偶尔晃动10分钟，在室温下，用1％的甲醛。 </li><li>淬火通过加入终浓度为125 mM的甘氨酸与孵育5分钟，在室温下交联。 </li><li>洗涤细胞用1X磷酸盐缓冲盐水（PBS）两次，倾析PBS中，然后加入0.2毫升的PBS。 </li><li>收集细?...

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开展全基因组的蛋白-DNA相互作用协会使用的ChIP-seq的档案现在是可行的，因为已经非常最近展示了与其他转录因子2,3。一个成功的测序结果的关键是一个高品质的染色质免疫沉淀DNA模板的生成。 一旦DNA模板已生成并确定要充分丰富，可以把它在后续测序文库制备。例如，可以使用由供应商提供，Illumina公司的测序文库协议。这个库的大小选择，可以通过凝胶电泳和随?...

对于许多年一直存在的未满足的需要，以确定约束和受给定的蛋白质的基因组范围，特别是那些在转录因子类基因组。 奥多姆等[1]用染色质免疫沉淀技术（ChIP）结合系统识别占用由预先指定的转录调控因子，在人体的肝脏和胰岛基因的启动子微阵列。随后，约翰逊等人开发了大规模染色质免疫沉淀的基础上直接超高通量DNA测序技术（ChIP-seq的）为了全面映射整个哺乳动物基因组的蛋白-DNA相互作用。测试的情况下，他们在体内的结合映射神经元限制性沉默因子（NRSF）到1946年人类基因组中的位置。数据显示清晰分辨率装订位置（50个碱基对），这有利于双方的i染料溶液的图案和NRSF结合基序识别。这些芯片-Seq数据也有较高的敏感性和特异性和统计的信心（P &lt;10-4），推断新的候选相互作用是重要的属性。 Robertson 等人。3也可用于芯片起，以映射STAT1目标干扰素-γ（IFN-γ）刺激和未刺激的人HeLa S3细胞在体内 。芯片起，使用15.1和1290万唯一对应的序列读取和估计错误​​发现率小于0.001，他们确定了41,582和11,004公认的STAT1结合区在刺激和未刺激细胞，分别。已知含有STAT1干扰素反应的结合位点4-8的34个位点，芯片起发现有24人（71％）。芯片的seq目标富集序列与已知的STAT1结合基序。比较两个现有的芯片-PCR数据集建议该芯片起的灵敏度为70％和92％，特异度为至少95％之间。此外，它的ChIP-seq的很清楚，同时提供低复杂性和敏感性分析，随着测序深度。 由于这样，“下一代”的基因组测序技术提供了1-2个数量级的增加量的序列，可以产生成本效益比老技术9。因此，芯片的seq方法直接提供的全基因组覆盖率哺乳动物蛋白-DNA相互作用进行有效的分析。 在2006年，强烈关联，在7-2（TCF7L2）与2型糖尿病的基因转录因子的变种被发现10。其他研究者已经独立复制这一发现在不同的种族，有趣的是，从2型糖尿病全基因组关联研究发表在自然 11,12 </sTCF7L2高达13-15， 科学和其他地方的16,17，的确是最强的协会，这是目前被认为是最重要的2型糖尿病的遗传发现迄今18-20。此外，TCF7L2一直与癌症风险21,22，事实上，这方面变得更加明显8q24染色体位点，揭示了全基因组关联研究，许多癌症，包括大肠癌，被证明是由于极端的上游TCF7L2结合元件驱动MYC基因的转录23,24。因此，有极大的兴趣，确定下游受这个关键转录因子的基因。 基于与TCF7L2作为该方法的一个例子的经验，本文概述了如何产生高品质的芯片的DNA模板。大肠癌细胞株HCT116芯片，为了建立一个高甲阶酚醛树脂为后续测序ution地图基因TCF7L2 25约束的努力产生进一步了解其复杂性状的发病机制中的关键作用。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td></td>
 </tr>
 <tr>
 <td>QIAquick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28104 </td>
 <td></td>
 </tr>
 <tr>
 <td>EZ-ChIP Kit </td>
 <td>Millipore </td>
 <td>17-371 </td>
 <td></td>
 </tr>
 <tr>
 <td>GoTaq Hot Start Polymerase </td>
 <td>Promega</td>
 <td>M5001</td>
 <td></td>
 </tr>
 <tr>
 <td>Misonix Sonicator</td>
 <td>Qsonica </td>
 <td>XL-2000 </td>
 <td></td>
 </tr>
 <tr>
 <td>
 NanoDrop 1000 Spectrophotometer </td>
 <td>
 Thermo-Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Positive control primer sequences (TCF7L2-1) 
 Forward- 5'-TCGCCCTGTCAATAATCTCC-3' 
 Reverse- 5'-GCTCACCTCCTGTATCTTCG-3' 
 Negative control primer sequences (CTRL-1) 
 Forward-5'-ATGTGGTGTGGCTGTGATGGGAAC-3'
 
 Reverse- 5'-CGAGCAATCGGTAAATAGGTCTGG-3'</td>
 </tr>
 </tbody>
</table>

generation of high quality chromatin immunoprecipitation dna template for high-throughput sequencing (chip-seq)

芯片测序技术（ChIP-seq的）方法直接提供全基因组的覆盖面，结合染色质免疫沉淀技术（ChIP）和大规模并行测序可以用来识别剧目哺乳动物的DNA序列转录因子在体内的约束。 “下一代”的基因组测序技术提供了1-2个数量级的增加量序列，可以是成本有效的ChIP-seq的方法直接进行有效的分析哺乳动物全基因组覆盖率较老的技术，从而使生成的蛋白质-DNA相互作用。 对于成功的ChIP-seq的方法，必须产生高品质芯片的DNA为模板，以获得最佳的测序成果。的说明是基于经验，最强烈暗示发病机制中的2型糖尿病，即转录因子的转录因子7类2（TCF7L2基因的蛋白产物）。在各种癌症中，这一因素也被牵连。 概述是如何产生的，以高品质的芯片DNA模板来自大肠癌细胞株HCT116通过测序，以确定约束TCF7L2基因，进一步了解其发病机制中的关键作用，建立一个高解析度地图复杂性状。

已被超声处理的染色质，并用RNA酶和蛋白酶处理后，将样品在2％琼脂糖凝胶上运行的应出示涂抹标本所需的大小的DNA与散装。如果几个不同的周期进行测试，逐渐减少的周期数增加（ 图2）的大小应被看作是。 完成后的协议的免疫沉淀部的富集，可以由PCR或实时PCR检查。 PCR样品在琼脂糖​​凝胶上运行，应该有输入和芯片乐队（使用抗体蛋白的兴趣，这是在这?...

Watch this Scientific Journal Video about Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq at JoVE.com

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

染色质免疫沉淀和超高通量测序（芯片起）的组合可以识别并映射在一个给定的组织或细胞系的蛋白质-DNA相互作用。概述是如何产生一个高质量的ChIP随后的测序模板，使用的经验，例如转录因子TCF7L2。

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 ...

Research

JoVE Journal

Biology

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

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

科研

生物学

Targeted DNA Methylation Analysis by Next-generation Sequencing

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

平行高通量单分子动力学测定，用于现场特异性DNA裂解

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

改进的高通量DNA提取板加载方法，降低污染的可能性

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...