Core B: Single Cell Protein and RNA Sequencing Core

核心 B：单细胞蛋白质和 RNA 测序核心

• 批准号: 10334092
• 负责人: Klaus F. Ley
• 金额: $ 5.35万
• 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 2022-06-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10334092
• 关键词: ATAC-seq; Antibodies; Atherosclerosis; B-Lymphocytes; B-cell receptor repertoire sequencing; Binding; Bioinformatics; Biostatistics Core; Cardiovascular system; Cell Communication; Cells; Clinical; Cytometry; DNA Sequencing Facility; Data; Epidemiologist; Excision; Experimental Designs; Genomics; Guidelines; Human; Immune; Leukocytes; Light; Manuscripts; Meta-Analysis; Mus; Oligonucleotides; Peptide Sequence Determination; Peripheral Blood Mononuclear Cell; Phenotype; Postdoctoral Fellow; Progress Reports; Publishing; Quality Control; Research; Running; Sampling; Services; Shipping; Signal Transduction; T cell receptor repertoire sequencing; T-Lymphocyte; Tissues; Visualization; Work; base; cohort; computerized data processing; data quality; design; experience; human data; improved; macrophage; monocyte; single cell proteins; single-cell RNA sequencing; transcriptome sequencing

Core B Summary Core B serves all 4 projects in this PPG for all RNA sequencing needs, including bulk and single cell RNA- sequencing (scRNA-Seq), protein sequencing by oligonucleotide-tagged antibodies (Ab-Seq), T- and B-cell receptor sequencing (TCR-Seq and BCR-Seq) and ATAC-Seq. Core B is responsible for all quality controls along the workflow. Core B staff consists of wet lab staff and bioinformaticians. They interact with postdocs, technicians, epidemiologists and PIs from the Human Clinical Cardiovascular and Biostatistics Core (Clinical Core C) and from each of the four projects. Workflows are established for mouse (macrophages, sorted T cells) and human (PBMC) cells. Hashtags and antibodies are fully validated, including thresholding, which improves data quality by eliminating signal from unbound antibody and non-specific antibody binding. The previous version of this core (core E in the 2016 Hedrick PPG) was the first group world-wide to use scRNA- Seq in atherosclerosis research. We established and published guidelines for workflows and quality controls in scRNA-Seq, batch effect, doublet and dead cell removal. Core B provides wet lab and bioinformatics service plus well-developed interfaces with all 4 projects and core C. For Ab-Seq, we validated a 192 oligonucleotide- tagged human antibody panel (Biolegend), run on 10x Genomics 5’, which will be used for Ab-Seq in most of the proposed human studies. For mice, we use the Biolegend 138 mouse antibody panel with 10x Genomics 5’. Using this platform, we have successfully assembled tens of thousands of TCRα and β chain pairs (scTCR- Seq) and BCR heavy and light chain pairs (scBCR-Seq). All 4 projects use core B. The specific aims are: (1) To provide the highest quality scRNA-Seq, scAb-Seq, scATAC-Seq, scTCR-Seq and scBCR-Seq for mouse and human samples, including fully documented and rigorous quality controls, designing and optimizing antibody panels and advice on all bioinformatics issues. (2) To provide the highest quality bulk RNA-Seq and ATAC-Seq for mouse and human samples, including fully documented and rigorous quality controls and support for experimental design and planning. (3) To effectively and seamlessly interface with postdocs, technicians, epidemiologists and PIs from the Clinical Core C and from each of the four projects, including sample shipping, receiving, thawing, data processing, visualization and delivery. To provide these services, we have built a team of wet lab technicians, bioinformaticians and computational biologists. Interfaces with all projects and core C are established and working well.

核心B总结 核心B服务于本PPG中的所有4个项目，满足所有RNA测序需求，包括批量和单细胞RNA- 测序（scRNA-Seq），通过标记抗体的蛋白质测序（Ab-Seq），T细胞和B细胞 受体测序（TCR-Seq和BCR-Seq）和ATAC-Seq。核心B负责所有质量控制 沿着工作流程。核心B人员包括湿实验室人员和生物信息学家。他们与博士后互动， 来自人类临床心血管和生物统计学核心（临床 核心C）和四个项目中的每一个。建立小鼠（巨噬细胞，分选T细胞）的工作流程。 细胞）和人（PBMC）细胞。主题标签和抗体经过充分验证，包括阈值处理， 通过消除来自未结合抗体和非特异性抗体结合的信号来提高数据质量。的 该核心的先前版本（2016年赫德里克PPG中的核心E）是全球第一个使用scRNA的小组， 动脉粥样硬化研究中的序列分析。我们制定并发布了工作流程和质量控制指南， scRNA-Seq、批量效应、双联体和死细胞去除。核心B提供湿实验室和生物信息学服务 加上与所有4个项目和核心C的良好接口。对于Ab-Seq，我们验证了192个寡核苷酸- 标记的人抗体组（Biolegend），在10 x Genomics 5 '上运行，其将用于大多数实验中的Ab-Seq。 拟议的人类研究。对于小鼠，我们使用Biolegend 138小鼠抗体组和10 x Genomics 5'.利用这个平台，我们已经成功地组装了数万个TCRα和β链对（scTCR-β）。 Seq）和BCR重链和轻链对（scBCR-Seq）。所有4个项目都使用核心B。具体目标是：（1） 为小鼠提供最高质量的scRNA-Seq、scAb-Seq、scATAC-Seq、scTCR-Seq和scBCR-Seq 和人体样本，包括完整记录和严格的质量控制，设计和优化 抗体面板和所有生物信息学问题的建议。(2)为了提供最高质量的批量RNA-Seq， 用于小鼠和人类样本的ATAC-Seq，包括完整记录和严格的质量控制， 支持实验设计和规划。(3)为了有效地与博士后无缝对接， 来自临床核心C和四个项目的技术人员、流行病学家和PI，包括 样品运输、接收、解冻、数据处理、可视化和交付。为了提供这些服务，我们 已经建立了一个由湿实验室技术人员、生物信息学家和计算生物学家组成的团队。与所有 项目和核心C已经建立并运行良好。