Internally Calibrated Chromatin Immunoprecipitation Using Barcoded Nucleosomes

 描述（申请人提供）：染色质免疫沉淀（ChIP）是研究基因表达表观遗传调控的核心方法之一。分析方法，如qPCR，微阵列和下一代测序（NGS）增加了通量，但没有ChIP数据集的可靠性。使用最先进的试剂，ChIP最好是半定量的，最坏的是不能代表体内翻译后修饰（PTM）密度。为了解决这一不足，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，用于随后的表观遗传标记的标准化和定量。 该方法，这里称为内部校准免疫沉淀测序，或ICeChIP-Seq，最近由EpiCypher，Inc.独家授权。并依赖于EpiCypher和Ruthenburg实验室合作开发和制造的设计师核小体。这些核小体用感兴趣的表观遗传标记修饰，并包裹在含有独特的识别条形码序列的DNA序列中。将条形码化的设计者核小体以各种浓度添加到ChIP反应中，其含有纯化的染色质和针对感兴趣的表观遗传标记的珠粒附着的下拉抗体。免疫沉淀后，分析下一代测序数据中检测到的1）每个条形码和2）免疫沉淀的输入DNA的读段数，所得比率用于定量计算IP富集。然后可以将读数归一化为每个条形码化核小体的输入浓度，提供用于定量样品DNA读数的标准曲线。带条形码的核小体可用作校准品，因为它们与样品染色质在ChIP反应期间经历的变异性来源相同，并且它们与IP的靶标精确相似。该检测的概念验证开发集中在以下方面：条形码化核小体的合成;其质量分析; H3 K4 me 3检测的开发;分析算法;表征信噪比;最小染色质输入需求和日常变异性。鉴于迄今为止产生的强大数据，以及对改进ChIP-Seq检测的迫切需求，我们提出了一项直接进入第二阶段的工作计划，以实现这种强大方法的商业化。第一阶段报告提供了令人信服的证据，证明该方法是对现有方法的巨大改进，第二阶段的目标集中在制造H3 K4 me 3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。

 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.