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(申请人提供)：在下一代测序和表观基因组学的帮助下，对启动子和增强子等调控元件的鉴定进展迅速。可以使用两种不同的组蛋白修饰来识别和区分这些元件。这涉及组蛋白H3在赖氨酸4上的甲基化状态。三甲基化(H3K4me3)和单甲基化(H3K4me1)分别唯一地定位于启动子和增强子。使用CHIP-SEQ，染色质免疫沉淀与下一代测序相结合，使用针对这些修饰的抗体，使人们能够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有的方式标记这些元件，特别是在带有H3K4me1的增强剂的情况下。因此，如果我们想要有一份这些调控因素的全面清单，将需要从身体的大多数细胞类型构建地图。这是一个主要的障碍。对于CHIP-SEQ的目的，最常见的是对从10^8到10^6的大量细胞进行CHIP。对于大多数细胞类型来说，这种规模是达不到的，特别是如果目标是从细胞系转移到主要细胞类型。事实证明，在不降低CHIP-SEQ规模的情况下，继续我们目前确定此类关键监管要素的速度将是具有挑战性的。最近，已经进行了许多尝试来缩减文库扩增的输入材料，较少关注小规模芯片，更少关注小规模染色质碎裂。我们正在提出一种方法，有可行的替代方案，我们相信这种方法将把芯片序列的规模减少2-4个数量级，可能低至1-100个电池。该方法考虑了从染色质碎裂到文库扩增的整个过程。我们的方法很大程度上得益于使用体外转座在芯片之前向染色质添加测序适配器。最终，这种方法可能会帮助我们实现单细胞分析。 与公共卫生相关：我们正在开发一种方法，以帮助识别功能性调控元素，如增强剂和促进剂。我们的方法是将芯片序列所需的输入材料的规模减少几个数量级。该方法减少了当前在库准备期间所需的几个步骤，这可以提供降低成本的附加好处。