Core B

核心B

• 批准号: 10428166
• 负责人: Christopher D Scharer
• 金额: $ 46.38万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-25 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10428166
• 关键词: Address; B-Lymphocytes; Bioinformatics; Biological Assay; CRISPR/Cas technology; Cell Maintenance; Cell physiology; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Chromatin; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; Computer Analysis; Cost Savings; DNA; DNA Methylation; Data; Data Analyses; Data Set; Data Storage and Retrieval; Disease; Ensure; Epigenetic Process; Experimental Designs; Flow Cytometry; Funding; Gene Expression Profile; Generations; Genes; Genetic; Genetic Transcription; Genome; Genome engineering; Genomic Library; Goals; Health; Histones; Human; Infection; Knock-out; Libraries; Maintenance; Manuscripts; Maps; Molecular; Molecular Biology; Mus; Online Systems; Pathway interactions; Plasma Cells; Plasmids; Post-Translational Protein Processing; Preparation; Process; Protocols documentation; Publications; Quality Control; Research Personnel; Resources; Sampling; Series; Services; Sorting - Cell Movement; Standardization; Structure; Technology; Testing; Transcript; Transposase; Viral; Work; base; bisulfite sequencing; cell type; data analysis pipeline; data exploration; data quality; data sharing; data submission; deep sequencing; design and construction; epigenomics; experimental study; genetic manipulation; genome-wide; genomic data; genomic platform; histone modification; interactive tool; interest; member; methylation pattern; overexpression; plasma cell development; plasma cell differentiation; programs; public repository; repository; single-cell RNA sequencing; success; synergism; tool; transcriptome; transcriptome sequencing; vector; whole genome

The projects within this PPG proposes aims that will determine the transcriptional and epigenetic programs of plasma cell development and maintenance. Additionally, the projects propose to use genome engineering to genetically manipulate B cells using CRISPR and over express cDNAs. To provide this expertise, ensure standardized protocols, integration of results and their subsequent analyses, and the sharing of data, the creation of an Epigenomics, Bioinformatics, and Genome Engineering Core (Core B) within this PPG is proposed. Core B will provide state-of-the-art technologies, molecular biology expertise, and bioinformatic services that assess DNA methylation, chromatin state and accessibility, and transcript expression through deep sequencing of both bulk and single-cell datasets. To support the genome engineering experiments Core B will identify functional sgRNA, provide cloning and viral preparation services, maintain plasmid repository and protocols supporting B cell genome engineering. To serve the projects, three Aims are proposed. Aim 1. Provide uniform and quality library preparation and sequencing to determine the transcriptome, DNA methylation patterns, chromatin accessibility, and histone modifications. Core B will create high-quality libraries and facilitate deep sequencing based on five technologies to derive epigenetic programming. RNA-seq will be used to determine the transcriptome. Reduced Representation Bisulfite Sequencing (RRBS) or Whole Genome Bisulfite Sequencing (WGBS) will be used to assess DNA methylation. The Assay for Transposase Accessible Chromatin (ATAC-seq) will determine chromatin accessibility. Cleavage Under Targets and Tagmentation (CUT&Tag) will be used to determine histone posttranslational modifications. Aim 2. Provide iterative bioinformatic computational analysis of datasets. A question driven, iterative bioinformatics analysis will be used to derive and examine the molecular programming of B cells and plasma cells. Core B will draw upon considerable expertise in both single-cell and bulk B cell/plasma cell genomic datasets with the capability of integrating data across platforms, disease and conditions, and species. Core B will provide long-term data storage, and facilitate sharing of processed datasets, including the use of interactive data exploration tools to facilitate data analysis by the entire program. Aim 3. Provide a B cell genome engineering platform using CRISPR/Cas9 and cDNA overexpression. Core B will test sgRNAs to identify those that provide maximal deletion, clone sgRNAs of interest into viral-based vectors and prepare stocks, and provide optimized protocols for infection and ultimately genome engineering of B cells. Additionally, Core B will maintain a centralized repository of vectors that contain flow cytometry compatible markers for sorting and selection, genome-wide sgRNA pools, and constructs that allow overexpression of cDNAs. Thus, Core B will provide a common library preparation and genome engineering resource and analytical platform that will serve to facilitate the success of each of the projects.

该 PPG 内的项目提出的目标将确定转录和表观遗传程序 浆细胞的发育和维持。此外，这些项目建议利用基因组工程来 使用 CRISPR 和过度表达 cDNA 对 B 细胞进行基因操作。为了提供这种专业知识，请确保 标准化协议、结果整合及其后续分析、数据共享、创建 提议在该 PPG 中建立表观基因组学、生物信息学和基因组工程核心（核心 B）。核 B 将提供最先进的技术、分子生物学专业知识和评估生物信息学服务 DNA 甲基化、染色质状态和可及性，以及通过深度测序的转录表达 批量和单细胞数据集。为了支持基因组工程实验，Core B 将鉴定功能 sgRNA，提供克隆和病毒制备服务，维护质粒库和支持B的协议 细胞基因组工程。为了服务于这些项目，提出了三个目标。目标 1. 提供统一和高质量 文库制备和测序以确定转录组、DNA 甲基化模式、 染色质可及性和组蛋白修饰。核心 B 将创建高质量的库并促进 基于五种技术的深度测序推导表观遗传编程。 RNA-seq 将用于 确定转录组。简化代表性亚硫酸氢盐测序 (RRBS) 或全基因组亚硫酸氢盐 测序 (WGBS) 将用于评估 DNA 甲基化。转座酶可及染色质的测定 (ATAC-seq) 将确定染色质可及性。目标下的切割和标记 (CUT&Tag) 将 用于确定组蛋白翻译后修饰。目标 2. 提供迭代生物信息学 数据集的计算分析。将使用问题驱动的迭代生物信息学分析来得出 并检查 B 细胞和浆细胞的分子编程。核心B将利用大量 拥有单细胞和批量 B 细胞/浆细胞基因组数据集方面的专业知识，并具有整合数据的能力 跨平台、疾病和条件以及物种。 Core B将提供长期数据存储，并方便 共享处理后的数据集，包括使用交互式数据探索工具来促进数据分析 通过整个程序。目标 3. 提供使用 CRISPR/Cas9 和 cDNA 的 B 细胞基因组工程平台 过度表达。核心 B 将测试 sgRNA，以识别那些提供最大删除、克隆 sgRNA 的 sgRNA。 对基于病毒的载体感兴趣并准备库存，并提供优化的感染方案，最终 B 细胞的基因组工程。此外，Core B 将维护一个集中的向量存储库，其中包含 用于排序和选择的流式细胞术兼容标记、全基因组 sgRNA 库以及构建体 允许 cDNA 过度表达。因此，Core B将提供通用的文库制备和基因组 工程资源和分析平台将有助于促进每个项目的成功。