Core E: Cell sorting, CyTOF and RNA-Seq

核心 E：细胞分选、CyTOF 和 RNA-Seq

• 批准号: 10188604
• 负责人: Klaus F. Ley
• 金额: $ 54.45万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188604
• 关键词: Aliquot; Alleles; Antibodies; Antigen-Presenting Cells; Antigens; Arterial Fatty Streak; Arteries; Atherosclerosis; B-Lymphocytes; BLR1 gene; Bioinformatics; Biological; Bone Marrow; CD4 Positive T Lymphocytes; CXCR4 gene; California; Cell Communication; Cell Separation; Cell surface; Cells; Cytometry; DNA; Data; Data Analyses; Dimensions; Ensure; Flow Cytometry; Freezing; Genes; Genome; Genomics; Greater sac of peritoneum; HLA Antigens; Hand; Harvest; Human; Immune; In Vitro; Individual; Institutes; Lanthanoid Series Elements; Leukocytes; Libraries; Liquid substance; Maps; Memory; Methods; Modernization; Monoclonal Antibodies; Mus; National Heart, Lung, and Blood Institute; Nitrogen; Nucleotides; Pathway Analysis; Patients; Performance; Peripheral Blood Mononuclear Cell; Peritoneal; Phenotype; Plasma; Population; Preparation; Principal Component Analysis; Proteins; Quality Control; RNA; Rare Earth Metals; Running; Sampling; Shipping; Ships; Spleen; T-Lymphocyte; Technology; Time; Tissues; Trans-Omics for Precision Medicine; Tube; Universities; Virginia; antibody conjugate; base; cell type; data quality; design; differential expression; dimensional analysis; experience; experimental study; genome-wide; high dimensionality; human RNA sequencing; human data; human leukocyte antigen testing; in vivo; instrument; macrophage; mass spectrometer; monocyte; novel; preservation; terminally differentiated effector memory (TEM) T cells; tool; transcription factor; transcriptome; transcriptome sequencing; trizol; vapor

Core E description Core E has four functions, serving all 4 projects. First, human and mouse T cells, B cells and monocytes will be sorted from peripheral blood mononuclear cells (PBMCs) and sorted into trizol, RNA extracted, quality controls (bioanalyzer, Agilent TapeStation) performed, libraries prepared and RNA-Seq done to obtain ~50-70 million Illumina reads per sample. Preliminary data show excellent sequencing depth and data quality even in long- term frozen PBMCs. Mouse and human samples are focused on monocyte subsets for project 1, treated human and sorted mouse macrophages for project 2, B1 cells from peritoneal cavity, spleen and bone marrow in wild-type, Cxcr4-/- and Cxcr5-/- mice for project 3, and dextramerhigh, dextramerlow, dextramernegative effector memory (TEM) cells and naïve CD4 T cells for project 4. Second, 36 to 42-parameter mass cytometry (CyTOF) followed by high-dimensional analysis (SPADE, viSNE) will be used for unsupervised clustering. We have already acquired or conjugated, titrated and validated more than 100 monoclonal antibodies, developed one human panel that will serve all projects and four human panels that serve each project individually. We will use about 1 million cells from each tube for this, and the remaining about 7 million cells will be used for RNA-Seq. We also will run CyTOF on mouse. CyTOF identifies the known cell types and likely will suggest new, unknown cell types (new subsets of monocytes, B cells, T cells). Third, sorted T cells and antigen presenting cells (APCs) will be distributed to project 4 and sorted monocytes to project 1 for functional experiments in vivo and in vitro as detailed in the projects. Finally, core E will also provide basic bioinformatics support. This includes post-sequencing quality controls for RNA-Seq, mapping reads, differential expression (DE), heat maps, Venn diagrams and principal component analysis (PCA).

核心E描述 核心E具有四个功能，可为所有4个项目提供服务。首先，人和小鼠T细胞，B细胞和单核细胞将是 从外周血单核细胞（PBMC）分类，并分类为Trizol，提取RNA，质量控制 （生物分析仪，敏捷式挂毯）执行，准备的库和RNA-seq进行了约500-70万 Illumina读取每个样品。初步数据显示了出色的测序深度和数据质量，即使 术语冷冻PBMC。小鼠和人类样品专注于项目1的单核细胞子集 项目2的人类和分类的小鼠巨噬细胞，腹膜腔，sle骨和骨髓的B1细胞 在野生型中，cxcr4 - / - 和cxcr5 - / - 项目3的小鼠，以及右旋烷烃，右旋烷烃，dextramernegative效应器 项目4的记忆（TEM）细胞和幼稚的CD4 T细胞。 然后进行高维分析（Spade，Visne），将用于无监督的聚类。我们有 已经获得或共轭，滴定和验证了100多种单克隆抗体，开发了一种 将为所有项目和四个人类面板提供服务的人体面板，可分别为每个项目提供服务。我们将使用 为此，每个管中约有100万个细胞，其余约700万个细胞将用于RNA-Seq。 我们还将在鼠标上运行cytof。 cytof识别已知的细胞类型，可能会暗示新的，未知的 细胞类型（单核细胞，B细胞，T细胞的新子集）。第三，分类的T细胞和抗原呈现细胞 （APC）将分配给项目4，并将单核细胞分配给项目1，以进行体内功能实验和 在项目中详细介绍的体外。最后，核心E还将提供基本的生物信息学支持。这包括 RNA-seq，映射读数，差分表达式（DE），热图，Venn的后期质量控制 图和主成分分析（PCA）。