Core B

该PPG中的项目提出的目标将确定的转录和表观遗传程序

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.