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
基本信息
- 批准号:8463011
- 负责人:
- 金额:$ 19.31万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2012
- 资助国家:美国
- 起止时间:2012-04-24 至 2015-03-31
- 项目状态:已结题
- 来源:
- 关键词:AccountingAntibodiesBiochemical ReactionBiological AssayCell CountCell LineCellsChIP-on-chipChIP-seqChromatinCollectionCoupledDNADrosophila genusElementsEnhancersGenetic Enhancer ElementGenomeGoalsHistone H3HumanHuman GenomeIn VitroIndiumLibrariesLibrary MaterialsLigationLysineMapsMethodsMethylationModificationMono-SMusNoiseOrganismPreparationProcessRegulatory ElementReportingResearchSample SizeSignal TransductionSonicationTransposaseUnited States National Institutes of HealthWorkcell typechromatin immunoprecipitationcostcrosslinkepigenomeepigenomicsgenome-widehistone modificationmeetingsnext generation sequencingpromotersingle cell analysistranscription factor
项目摘要
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.
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,启动子和增强子等调控元件的鉴定正在快速进行。两种不同的组蛋白修饰可以用来识别和区分这些元素。这涉及赖氨酸4处组蛋白H3的甲基化状态。三甲基化(H3K4me3)和单甲基化(H3K4me1)分别定位于启动子和增强子。利用ChIP- seq,染色质免疫沉淀结合下一代测序,以及针对这些修饰的抗体,可以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的方式标记这些元素,特别是在H3K4me1增强子的情况下。因此,如果我们要有这些调控元素的全面列表,就需要从人体的大多数细胞类型中构建图谱。这里存在一个主要障碍。对于ChIP-seq, 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,这种规模是无法达到的,特别是如果目标是摆脱细胞系而专注于原代细胞类型。如果不减少ChIP-seq的规模,继续我们目前确定这些关键监管要素的步伐将是具有挑战性的。最近,已经进行了许多尝试,以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碎片化。我们正在提出一种可行的替代方法,我们相信它将把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断裂到文库扩增的整个过程。我们的方法很大程度上得益于在ChIP之前使用体外转位将测序适配器添加到染色质上。最终,这种方法可以帮助我们实现单细胞分析。
项目成果
期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Raymond David Hawkins其他文献
Raymond David Hawkins的其他文献
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{{ truncateString('Raymond David Hawkins', 18)}}的其他基金
Three-dimensional conformation changes associated with T cell memory and autoimmunity
与 T 细胞记忆和自身免疫相关的三维构象变化
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10115995 - 财政年份:2020
- 资助金额:
$ 19.31万 - 项目类别:
Three-dimensional conformation changes associated with T cell memory and autoimmunity
与 T 细胞记忆和自身免疫相关的三维构象变化
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10456277 - 财政年份:2020
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$ 19.31万 - 项目类别:
Three-dimensional conformation changes associated with T cell memory and autoimmunity
与 T 细胞记忆和自身免疫相关的三维构象变化
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10689314 - 财政年份:2020
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10264086 - 财政年份:2020
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Functional assessment of distal regulatory SNPs associated with type 1 diabetes.
与 1 型糖尿病相关的远端调节 SNP 的功能评估。
- 批准号:
9769721 - 财政年份:2015
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Functional assessment of distal regulatory SNPs associated with type 1 diabetes.
与 1 型糖尿病相关的远端调节 SNP 的功能评估。
- 批准号:
9118987 - 财政年份:2015
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Functional validation of Rheumatoid Arthritis-associated distal regulatory SNPs i
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8810054 - 财政年份:2015
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$ 19.31万 - 项目类别:
Functional assessment of distal regulatory SNPs associated with type 1 diabetes.
与 1 型糖尿病相关的远端调节 SNP 的功能评估。
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9934717 - 财政年份:2015
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Effects of PAHs on the Epigenome and Differentiation Capacity of Embryonic and Ne
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- 批准号:
8536291 - 财政年份:2012
- 资助金额:
$ 19.31万 - 项目类别:
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 - 财政年份:2012
- 资助金额:
$ 19.31万 - 项目类别:
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