Gene expression, Epigenetics and Bioinformatics Core

基因表达、表观遗传学和生物信息学核心

基本信息

批准号：
10683264
负责人：
Matthew Eugene Pipkin
金额：
$ 15.07万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10683264
关键词：
ATAC-seq Address Algorithms Award B-Lymphocytes Base Pairing Binding Binding Sites Bioinformatics Biological Assay CD8-Positive T-Lymphocytes Cell Count Cell physiology Cells Cellular Immunity ChIP-seq Chromatin Chromatin Structure Cloning Clustered Regularly Interspaced Short Palindromic Repeats Computer Analysis Core Facility Creativeness DNA DNA Polymerase II DNA-Protein Interaction Data Data Analyses Dedications Doctor of Philosophy Epigenetic Process Event Evolution Faculty Gene Expression Gene Expression Profiling Genes Genetic Transcription Genomics Goals Guide RNA Human Resources Immune response Individual Infection Laboratories Libraries Lymphocyte Maps Measures Memory Messenger RNA Methods Molecular Nucleic Acid Regulatory Sequences Nucleosomes RNA RNA Interference RNA Sequences RNA interference screen RNA library Recording of previous events Regulation Research Institute Resolution Sampling Services Standardization System T cell differentiation T cell regulation T-Lymphocyte Subsets Transposase bioinformatics pipeline chromatin immunoprecipitation chromatin remodeling data management effector T cell experience experimental study gene regulatory network genome-wide genomic data histone modification in vivo innovation instrument large datasets loss of function mRNA Expression next generation sequencing programs tool transcription factor transcriptome sequencing tumor

项目摘要

PROJECT SUMMARY / ABSTRACT Core C (Pipkin) The overarching goal of Core C is to provide standardized, uniform and innovative next generation sequencing (NGS) and computational approaches to address the genome-scale experiments proposed in Projects 1-3. The individual Projects goals are to analyze pooled in vivo screens, gene expression, transcription and chromatin structure and to discern the basic molecular regulation of T and B cell mediated immunity. Core C will facilitate these objectives by using NGS approaches to analyze (1) pooled in vivo gene loss-of-function screens by sequencing, (2) expression of chromatin-associated RNA (nascent RNA), and mature mRNA from limiting ex vivo cell numbers, and single cells (scRNA-seq) using RNA-seq; (3) chromatin accessibility via the assay for transposase-accessible chromatin followed by sequencing (ATAC-seq), and nucleosome organization (MNase- seq and BEM-seq); and (4) CRF and TF binding to near base-pair resolution after chromatin immunoprecipitation from small numbers of primary lymphocytes using ChIP-exo (Aims 1 and 2). Core C has demonstrated experience in developing and applying all of these approaches. In addition, Core C has established a centralized, interlinked and documented framework for the storage, analysis and sharing of these large datasets between Projects 1-3 (Aim 3), and will apply available algorithms in creative arrangements that comprise computational approaches to identify and define operational cis-regulatory regions based on chromatin accessibility, nucleosome organization and histone modifications, and to infer utilized TF binding site motifs within these regions and predict their cognate TFs. Furthermore, these observations will be correlated with transcriptional activity (nascent RNA expression) and RNA Pol II activity (ChIP-seq) and TF and CRF binding events (ChIP- exo), and overall gene expression (mRNA) in the context of gene-perturbations to clarify functionally how gene regulatory networks drive T cell differentiation and function during immune responses in vivo.

项目摘要 /摘要核心C（Pipkin）核心C的总体目标是提供标准化，统一和创新的下一代测序（NGS）和计算方法，以解决项目1-3中提出的基因组规模实验。这各个项目的目标是分析体内筛选，基因表达，转录和染色质的合并结构并辨别T和B细胞介导的免疫的基本分子调节。核心将有助于这些目标通过使用NGS方法分析（1）在体内基因丧失功能丧失屏幕中通过测序，（2）染色质相关的RNA（新生RNA）的表达和限制Ex的成熟mRNA 使用RNA-seq的体内细胞数和单细胞（SCRNA-SEQ）；（3）通过测定的染色质访问性转座酶可访问的染色质，然后进行测序（ATAC-SEQ）和核小体组织（MNase- seq和bem-seq）; （4）染色质免疫沉淀后的CRF和TF结合与接近碱基对分辨率从少量的主要淋巴细胞使用CHIP-EXO（目标1和2）。 C核心已证明在开发和应用所有这些方法方面的经验。此外，Core C已经建立了一个集中式，与这些大数据集的存储，分析和共享的相互联系和记录的框架之间项目1-3（AIM 3），并将在构成计算的创意安排中应用可用算法基于染色质可及性识别和定义操作顺式调节区域的方法，核小体组织和组蛋白的修饰，并推断在这些内部使用的TF结合位点基序区域并预测其同源TF。此外，这些观察结果将与转录相关活性（新生的RNA表达）和RNA POL II活性（CHIP-SEQ）以及TF和CRF结合事件（芯片 - 在基因扰动的背景下，EXO）和总体基因表达（mRNA），以阐明基因如何在功能上调节网络在体内免疫反应期间驱动T细胞分化和功能。