Small-scale chromatin preparation and ChIP-seq aided by in vitro transposition

体外转座辅助的小规模染色质制备和 ChIP-seq

• 批准号: 8309590
• 负责人: Raymond David Hawkins
• 金额: $ 23.16万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-24 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8309590
• 关键词: Accounting; Antibodies; Biochemical Reaction; Biological Assay; Cell Count; Cell Line; Cells; ChIP-on-chip; ChIP-seq; Chromatin; Collection; Coupled; DNA; Drosophila genus; Elements; Enhancers; Genetic Enhancer Element; Genome; Goals; Histone H3; Human; Human Genome; In Vitro; Indium; Libraries; Library Materials; Ligation; Lysine; Maps; Methods; Methylation; Modification; Mono-S; Mus; Noise; Organism; Preparation; Process; Regulatory Element; Reporting; Research; Sample Size; Signal Transduction; Sonication; Transposase; United States National Institutes of Health; Work; cell type; chromatin immunoprecipitation; cost; crosslink; epigenomics; genome-wide; histone modification; meetings; next generation; promoter; single cell analysis; transcription factor

DESCRIPTION (provided by applicant): Aided by next-generation sequencing and epigenomics, the identification of regulatory elements such as promoters and enhancers is progressing at a rapid pace. Two distinct histone modifications can be used to identify and distinguish these elements. This involves the methylation states of histone H3 at lysine 4. Trimethylation (H3K4me3) and monomethylation (H3K4me1) are uniquely localized to promoters and enhancers respectively. Employing ChIP- seq, chromatin immunoprecipitation coupled with next-generation sequencing, with antibodies against these modifications allows one to construct genome-wide maps in a variety of species. However, these modifications often mark such elements in a cell-specific manner, especially in the case of enhancers with H3K4me1. Therefore, if we are to have a comprehensive list of these regulatory elements, maps will need to be constructed from most cell types of the body. Here in lies a major hurdle. For the purpose of ChIP-seq, ChIP is most commonly performed on a large number of cells, ranging from 10^8 to 10^6. This scale is unattainable for most cell types, especially if the goal is to mov away from cell lines and focus on primary cell types. Without reducing the scale of ChIP-seq, it will prove challenging to continue the pace at which we are currently identifying such key regulatory elements. Recently, a number of attempts have been made to scale down input material for the library amplification, with fewer focusing on small-scale ChIP, and even less on small-scale chromatin fragmentation. We are proposing a method, with viable alternatives, that we believe will reduce the scale of ChIP-seq by 2-4 orders of magnitude, possibly as low as 1-100 cells. This method takes into account the entire process from chromatin fragmentation to library amplification. Our approach is greatly aided by the use of in vitro transposition to add sequencing adaptors to chromatin prior to ChIP. Ultimately, this method may help us achieve single cell analysis. PUBLIC HEALTH RELEVANCE: We are developing a method to aid the identification of functional regulatory elements, such as enhancers and promoters. Our approach is to reduce the scale of input material required for ChIP-seq by several orders of magnitude. This method alleviates several steps currently required during library preparation, which may provide an added benefit of reducing cost.

描述（由申请人提供）：在下一代测序和表观基因组学的帮助下，调控元件如启动子和增强子的鉴定正在快速发展。两种不同的组蛋白修饰可用于鉴定和区分这些元件。这涉及组蛋白H3在赖氨酸4处的甲基化状态。三甲基化（H3 K4 me 3）和单甲基化（H3 K4 me 1）分别独特地定位于启动子和增强子。采用ChIP-seq，染色质免疫沉淀结合下一代测序，与针对这些修饰的抗体，允许在各种物种中构建全基因组图谱。然而，这些修饰通常以细胞特异性方式标记这些元件，特别是在具有H3 K4 me 1的增强子的情况下。因此，如果我们要有一个完整的清单，这些调控元件，地图将需要从大多数细胞类型的身体。这里存在一个主要障碍。为了ChIP-seq的目的，ChIP最常在大量细胞上进行，范围从10^8到10^6。对于大多数细胞类型来说，这个规模是无法达到的，特别是如果目标是远离细胞系并专注于原代细胞类型。在不减少ChIP-seq规模的情况下，继续我们目前确定这些关键监管要素的步伐将具有挑战性。最近，已经进行了许多尝试来按比例缩小文库扩增的输入材料，较少关注小规模ChIP，甚至更少关注小规模染色质片段化。我们正在提出一种方法，并有可行的替代方案，我们相信这将使ChIP-seq的规模减少2-4个数量级，可能低至1-100个细胞。该方法考虑了从染色质片段化到文库扩增的整个过程。我们的方法大大有助于使用体外转座，在ChIP之前将测序衔接子添加到染色质中。最终，这种方法可以帮助我们实现单细胞分析。 公共卫生相关性：我们正在开发一种方法来帮助识别功能性调节元件，如增强子和启动子。我们的方法是将ChIP-seq所需的输入材料的规模减少几个数量级。该方法简化了文库制备过程中目前所需的几个步骤，这可以提供降低成本的额外益处。