Core B

该PPG中的项目提出了将决定转录和表观遗传计划的目标 浆细胞的开发和维护。此外，这些项目建议使用基因组工程来 使用CRISPR对B细胞进行基因操作，并过度表达cDNA。要提供此专业知识，请确保 标准化协议、综合成果及其随后的分析以及共享数据、创建 建议在该PPG内建立表观基因组学、生物信息学和基因组工程核心(核心B)。堆芯 B将提供最先进的技术、分子生物学专业知识和生物信息服务，以评估 DNA甲基化、染色质状态和可及性，以及通过对两者进行深度测序的转录本表达 批量数据集和单元格数据集。为了支持基因组工程实验，核心B将识别功能 SgRNA，提供克隆和病毒制备服务，维护质粒库和支持B组蛋白的协议 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.提供统一和高质量的 文库准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将创建高质量的图书馆并促进 基于五种技术的深度测序，以派生表观遗传编程。RNA-seq将用于 确定转录组。降低代表性亚硫酸盐测序(RRBS)或全基因组亚硫酸盐 测序(WGBS)将用于评估DNA甲基化。转座酶可及染色质的检测 (ATAC-SEQ)将决定染色质的可及性。靶下切割和标记(切割和标记)将 用于确定组蛋白翻译后修饰。目标2.提供迭代的生物信息学 数据集的计算分析。将使用问题驱动、迭代的生物信息学分析来推导 并研究B细胞和浆细胞的分子编程。核心B将利用大量的 具备整合数据能力的单细胞和大宗B细胞/浆细胞基因组数据集方面的专业知识 跨越平台、疾病和状况以及物种。核心B将提供长期的数据存储，并促进 共享已处理的数据集，包括使用交互式数据探索工具促进数据分析 通过整个程序。目的3.利用CRISPR/Cas9和c DNA构建B细胞基因组工程平台 过度表达。核心B将测试sgRNAs以确定那些提供最大删除的sgRNAs，克隆sgRNAs 对基于病毒的载体感兴趣，并准备库存，并为感染和最终提供优化的方案 B细胞的基因组工程。此外，核心B将维护一个集中的载体存储库，其中包含 用于分类和选择的流式细胞术兼容标记、全基因组sgRNA池和构建 允许过度表达cDNA。因此，核心B将提供通用的文库准备和基因组 工程资源和分析平台，将有助于促进每个项目的成功。

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.