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).

Core E描述 核心E有四个功能，服务于所有四个项目。首先，人类和小鼠的T细胞、B细胞和单核细胞将被 从外周血单核细胞（PBMC）中分选并分选到Trizol中，提取RNA，质量控制 （生物分析仪，Agilent TapeStation），制备文库并进行RNA-Seq，以获得约5000 - 7000万 Illumina读取每个样品。初步数据显示，即使在长时间的测序中， 足月冷冻PBMC。小鼠和人类样本的重点是项目1的单核细胞亚群，经过处理 人和分选小鼠巨噬细胞从腹腔、脾和骨髓中投射2、B1细胞 在野生型、Cxcr 4-/-和Cxcr 5-/-小鼠中，对于项目3，和右旋糖酐高、右旋糖酐低、右旋糖酐阴性效应子 记忆（TEM）细胞和幼稚CD 4 T细胞用于项目4。第二，36至42参数质谱细胞仪（CyTOF） 随后的高维分析（SPADE，viSNE）将用于无监督聚类。我们有 已经获得或结合，滴定和验证了100多个单克隆抗体，开发了一个 一个为所有项目服务的人工小组和四个分别为每个项目服务的人工小组。我们将使用 每个试管中约有100万个细胞，其余约700万个细胞将用于RNA-Seq。 我们还将在小鼠上运行CyTOF。CyTOF识别了已知的细胞类型，并可能提示新的未知细胞类型。 细胞类型（单核细胞、B细胞、T细胞的新亚群）。第三，分选的T细胞和抗原呈递细胞 （APC）将被分配到项目4，并将分选的单核细胞分配到项目1，用于体内功能实验， 如项目中详细说明的那样。最后，核心E还将提供基本的生物信息学支持。这包括 RNA-Seq测序后质量控制、作图读数、差异表达（DE）、热图、Venn 图和主成分分析（PCA）。