Internally Calibrated Chromatin Immunoprecipitation Using Barcoded Nucleosomes

使用条形码核小体进行内部校准染色质免疫沉淀

• 批准号: 9045474
• 负责人: Zu-Wen Sun
• 金额: $ 67.66万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-15 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9045474
• 关键词: Address; Algorithms; Antibodies; Area; Biological Assay; Calibration; Cells; Chicago; Chromatin; Core Facility; DNA; DNA Sequence; Data; Data Set; Development; Diagnostic; Enzymes; Epigenetic Process; Evaluation; Exhibits; Gene Expression; Genomic Segment; Genomics; Histones; Immunoprecipitation; Letters; Licensing; Lysine; Malignant Neoplasms; Methods; Molecular; Noise; Nuclear Extract; Nucleic Acids; Nucleosomes; Paper; Phase; Post-Translational Protein Processing; Reaction; Reading; Reagent; Reporting; Reproducibility; Research; Research Institute; Research Personnel; Sampling; Signal Transduction; Site; Source; Universities; Validation; analytical method; assay development; base; chromatin immunoprecipitation; chromatin protein; clinical application; commercialization; density; epigenetic regulation; experience; improved; in vivo; interest; next generation sequencing; novel therapeutics; public health relevance; reconstitution; research study

 DESCRIPTION (provided by applicant): Chromatin immunoprecipitation (ChIP) is one of the core methods in the study of epigenetic regulation of gene expression. Analytical methods, such as qPCR, microarray and next generation sequencing (NGS) have increased throughput, but not the reliability of ChIP datasets. Using state-of-the-art reagents, ChIP is at best semi-quantitative, and at worst unrepresentative of in vivo post-translational modification (PTM) density. To address this short-coming, EpiCypher, Inc. has partnered with Dr. Alex Ruthenburg of the University of Chicago who has pioneered a method to internally-standardize ChIP experiments for subsequent normalization and quantitation of epigenetic marks. The method, referred to here as Internally Calibrated Immunoprecipitation Sequencing, or ICeChIP-Seq, was recently licensed on an exclusive basis by EpiCypher, Inc. and relies on designer nucleosomes collaboratively developed and manufactured by EpiCypher and the Ruthenburg lab. These nucleosomes are modified with an epigenetic mark of interest and wrapped in a DNA sequence containing a unique, identifying barcode sequence. Barcoded designer nucleosomes are added to the ChIP reaction at various concentrations, containing purified chromatin and a bead-attached pull-down antibody against the epigenetic mark of interest. After immunoprecipitation, next generation sequencing data is analyzed for the number of reads detected for 1) each barcode and 2) immunoprecipitated input DNA with the resulting ratio used to compute IP enrichment quantitatively. Read number can then be normalized to input concentration for each barcoded nucleosome, providing a standard curve for quantitation of sample DNA reads. The barcoded nucleosomes serve as calibrators because they are subjected to the same sources of variability the sample chromatin experiences during the ChIP reaction, and they precisely resemble the target of the IP. Proof-of-concept development of the assay has focused on the following: synthesis of barcoded nucleosomes; analysis of their quality; development of an H3K4me3 assay; analysis algorithms; characterizing signal-to-noise ratio; minimum chromatin input demands and day-to-day variability. Given the strong data generated to date, and the urgent need for an improved ChIP-Seq assay, we have proposed a Direct to Phase II workplan toward the commercialization of this powerful method. The Phase I report provides compelling evidence that the method is a dramatic improvement over existing methods, with Phase II aims focused on manufacturing a kit for the H3K4me3 assay reagents, external validation of this assay and expanding the menu of PTM-specific ICeChIP-Seq reagents to other epigenetic marks.

 描述（申请人提供）：染色质免疫沉淀（ChIP）是基因表达表观遗传调控研究的核心方法之一，qPCR、微阵列和下一代测序（NGS）等分析方法增加了通量，但没有增加通量。 ChIP 数据集的可靠性使用最先进的试剂，ChIP 充其量是半定量的，最坏的情况是不能代表体内翻译后修饰。为了解决这一缺陷，EpiCypher, Inc. 与芝加哥大学的 Alex Ruthenburg 博士合作，后者开创了一种内部标准化 ChIP 实验的方法，用于后续表观遗传标记的标准化和定量。 ，此处称为内部校准免疫沉淀测序或 ICeChIP-Seq，最近获得 EpiCypher, Inc. 的独家许可，并依赖于设计者由 EpiCypher 和 Ruthenburg 实验室合作开发和制造的核小体用感兴趣的表观遗传标记进行修饰，并包裹在包含独特的条形码设计核小体中，以不同的浓度添加到 ChIP 反应中。纯化的染色质和针对感兴趣的表观遗传标记的珠子附着的下拉抗体在免疫沉淀后，分析下一代测序数据以检测检测到的读数数量。 1) 每个条形码和 2) 免疫沉淀的输入 DNA 以及用于定量计算 IP 富集的读数数量可以标准化为每个条形码核小体的输入浓度，从而提供用于定量样品 DNA 读数的标准曲线。作为校准品，因为它们在 ChIP 反应过程中受到样品染色质经历的相同变异源的影响，并且它们与 IP 的目标精确相似。该检测的概念验证开发侧重于以下方面：条形码核小体的合成；H3K4me3 分析算法的开发；表征最低染色质输入需求；鉴于迄今为止生成的强大数据以及改进 ChIP-Seq 检测的迫切需要，我们提出了一项直接进入 II 期工作计划，以实现这一强大方法的商业化。提供了令人信服的证据，表明该方法比现有方法有了显着改进，第二阶段的目标重点是制造 H3K4me3 检测试剂的试剂盒、该检测的外部验证以及将 PTM 特异性 ICeChIP-Seq 试剂的菜单扩展到其他表观遗传标记。