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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：染色质免疫沉淀（芯片）是ESSION研究的核心方法之一。芯片数据集。表观遗传标记。条形码序列的条形码设计器核小体是在不同浓度的Ntiboty中添加到碎屑核心的表观遗传标记，并分析了下一代测序数据，用于使用1个条形码的读数和2）。计算每个条形码核体的IP富集到输入浓度，将条形码作为校准剂，它们经历了相同的变异源。 HASS的概念集中在以下内容：核心体的核心核心;迫切需要改进的芯片 - 塞克测定法，我们已经直接向vidence进行了工作平台，即该方法是对现有方法H3K4me3测定试剂的巨大改进 - 其他表观遗传标记的特异性冰氮seq试剂。