Core B

本PPG中的项目提出了确定转录和表观遗传程序的目标， 浆细胞的发育和维持。此外，该项目还建议使用基因组工程， 使用CRISPR和过表达cDNA遗传操纵B细胞。要提供这种专业知识，请确保 标准化的协议，结果的整合及其随后的分析，以及数据的共享， 提出了在该PPG内的表观基因组学、生物信息学和基因组工程核心（核心B）。核心 B将提供最先进的技术、分子生物学专业知识和生物信息学服务， 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 批量数据集和单个单元格数据集。为了支持基因组工程实验，核心B将鉴定功能性 sgRNA，提供克隆和病毒制备服务，维护质粒库和支持B的方案 细胞基因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， 基于五种技术的深度测序，以获得表观遗传编程。RNA-seq将用于 确定转录组。还原型亚硫酸氢盐测序（RRBS）或全基因组亚硫酸氢盐测序 测序（WGBS）将用于评估DNA甲基化。转座酶可降解染色质的测定 （ATAC-seq）将确定染色质可及性。靶下切割和标签化（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推导 并检查B细胞和浆细胞的分子程序。核心B将利用相当多的 在单细胞和大量B细胞/浆细胞基因组数据集方面的专业知识，具有整合数据的能力 跨平台、疾病和条件以及物种。核心B将提供长期数据存储， 共享经处理的数据集，包括使用交互式数据探索工具，以便利数据分析 整个程序。目标3：利用CRISPR/Cas9和cDNA提供B细胞基因组工程平台 过度表达核心B将测试sgRNA以鉴定提供最大缺失的那些， 感兴趣病毒为基础的载体和制备股票，并提供优化的方案感染，并最终 B细胞的基因组工程。此外，核心B将维护一个集中的向量库，其中包含 用于分选和选择的流式细胞术相容标记物、全基因组sgRNA库和构建体， 允许cDNA的过表达。因此，核心B将提供共同的文库制备和基因组测序。 工程资源和分析平台，这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.