Gene expression, Epigenetics and Bioinformatics Core

项目总结/摘要 核心C（Pipkin） Core C的总体目标是提供标准化、统一和创新的下一代测序 (NGS)和计算方法来解决项目1-3中提出的基因组规模的实验。的 单个项目的目标是分析体内筛选、基因表达、转录和染色质 结构和辨别T和B细胞介导的免疫的基本分子调节。核心C将促进 这些目的通过使用NGS方法来分析（1）通过以下方法进行的合并的体内基因功能丧失筛选： 测序，（2）染色质相关RNA（新生RNA）的表达，以及来自限制性表达的成熟mRNA。 体内细胞数和使用RNA-seq的单细胞（scRNA-seq）;（3）通过测定的染色质可及性， 转座酶可及染色质测序（ATAC-seq）和核小体组织（MNase- seq和BEM-seq）;和（4）CRF和TF结合到染色质免疫沉淀后的近碱基对分辨率 使用ChIP-exo从少量的原代淋巴细胞中分离（目的1和2）。核心C已经证明 开发和应用所有这些方法的经验。此外，Core C还建立了一个集中的、 用于存储、分析和共享这些大型数据集的相互关联和有记录的框架， 项目1-3（目标3），并将在创造性的安排，包括计算 基于染色质可接近性鉴定和定义操作性顺式调节区的方法， 核小体组织和组蛋白修饰，并推断这些中利用的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RNA表达）和RNA Pol II活性（ChIP-seq）以及TF和CRF结合事件（ChIP-seq）。 exo）和基因扰动背景下的总体基因表达（mRNA），以阐明基因在功能上如何表达。 调节网络在体内免疫应答期间驱动T细胞分化和功能。

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.