Development of a method to multiplex ChIP-SEQ and ChIP-chip experiments

描述（由申请人提供）：我们研究的直接目标是生成一种方法，该方法将使研究人员能够在单次下一代DNA测序运行中进行多重染色质免疫荧光测序（ChIP-Seq）分析。在待开发的方法中：i）一组针对特异性DNA结合蛋白的抗体用DNA“ZipCode”独特地条形码化; ii）通过用甲醛处理细胞形成DNA结合蛋白和染色体DNA之间的共价交联; iii）使用对感兴趣的蛋白质具有特异性的DNA条形码化抗体组来选择性地共免疫沉淀蛋白质，共价交联的结合的DNA片段; iv）通过洗涤除去过量的抗体和染色体DNA; v）使用T4 DNA连接酶将富集的蛋白质结合的染色体DNA连接至抗体附着的ZipCode DNA，以及; vi）逆转免疫沉淀的蛋白质-DNA连接，并使用下一代测序仪测定回收的DNA，以确定由蛋白质和抗体结合的染色体DNA序列。识别DNA邮编 公共卫生相关性：研究人员将受益于对人类蛋白质功能的进一步了解。需要有方法来促进这种理解。我们研究的直接目标是产生一种方法，使研究人员能够在单个DNA测序运行中多重使用称为染色质免疫测序（ChIP-Seq）分析的方法。我们期望使用我们的高通量抗体发现管道来生产重组抗体，作为产生使这种多重方法成为可能所需的试剂的手段。

DESCRIPTION (provided by applicant): The immediate objective of our research is to generate a method that will enable researchers to multiplex Chromatin ImmunoPreciptation-Sequencing (ChIP-Seq) analysis in a single Next generation DNA sequencing run. In the to-be-developed method: i) a set of antibodies directed against specific DNA-binding proteins are uniquely bar-coded with a DNA 'ZipCode;' ii) covalent cross-links between DNA-binding proteins and chromosomal DNA are formed by treating cells with formaldehyde; iii) the set of DNA-barcoded antibodies specific to the proteins of interest are used to selectively coimmunoprecipitate the protein-bound DNA fragments that were covalently cross-linked; iv) excess antibodies and chromosomal DNAs are removed by washing; v) the enriched protein-bound chromosomal DNA is ligated to the antibody-attached ZipCode DNA using T4 DNA ligase, and; vi) the immunoprecipitated protein-DNA links are reversed and the recovered DNA is assayed using Next-generation sequencers to determine both the chromosomal DNA sequence bound by the protein and the antibody-identifying DNA ZipCode. PUBLIC HEALTH RELEVANCE: Researchers will benefit from an increased understanding of the function of human proteins. Methods are needed that can facilitate this understanding. The immediate objective of our research is to generate a method that will enable researchers to multiplex the method known as Chromatin ImmunoPreciptation-Sequencing (ChIP-Seq) analysis, in a single DNA sequencing run. We anticipate using our high-throughput antibody-discovery pipeline for producing recombinant antibodies as a means to generate the reagents needed to make this multiplexed method possible.