Ultra-Low and Single-Cell Transcriptome Analysis in Patient-Derived Rare CLL Precursor Cells

患者来源的罕见 CLL 前体细胞的超低和单细胞转录组分析

基本信息

批准号：
10012782
负责人：
Pearlly Yan
金额：
$ 14.9万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-20 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10012782
关键词：
Basic Science Biology Bone Marrow Buffers Cell Count Cell Separation Cell Volumes Cells Characteristics Clinical Communities DNA Methylation Data Drug resistance Epigenetic Process Foundations Genes Genome Genomics Genomics Shared Resource Hematology Individual Knowledge Libraries Malignant Neoplasms Manuscripts Medicine Mentors Nucleic Acids Patients Pharmaceutical Preparations Population Preparation Process Protocols documentation Publications RNA Research Research Assistant Research Design Research Personnel Sampling Standardization Technical Expertise Techniques Technology Time Translational Research Vision cancer recurrence cancer stem cell cell type college comparison group data quality design disorder later incidence prevention drug development drug sensitivity insight interest leukemic stem cell novel therapeutics operation personalized medicine precursor cell preservation professor single-cell RNA sequencing transcriptome transcriptomics

项目摘要

Project Summary/Abstract The Project PI is the Technical Director of the OSUCCC Genomics Shared Resources (GSR) and a Research Assistant Professor in the Division of Hematology at the OSU College of Medicine. She set up and directed the Illumina sequencing operation for close to 9 years now. As the GSR serves both the clinical/translational research community and the basic science communities at OSU, the Project PI has gained extensive insights related to the impact of quality (e.g., species, integrity, presence of contaminants, preservation process, nucleic acids extraction protocols) and quantity (concentration and total amount) of input material on sequencing library characteristics, sequencing data quality and read distributions in the genome. This wealth of knowledge helps to open up another front of research interest (in addition to DNA methylation and cancer epigenetics) for the PI resulting in the list of technology-related publications highlighted in the Biosketch document as well as the technology manuscripts under preparation as described in Research Strategy #3 and Research Strategy #5. For the R50 Application, the Mentor PIs (Dr. Muthusamy and Dr. Byrd) both share the same vision that properly controlled and well-designed low cell number- and single-cell RNA-seq in rare precursor populations in CLL and AML will allow us to evaluate the leukemic stem cell potential in CLL and AML, examine mechanism of drug sensitivity and biology in these rare cell populations. If successful, this research direction will open up avenues for novel therapies, relapse prevention and ultimately cellular level personalized medicine. As little is known about these precursor populations, careful characterization of their bulk transcriptomic profile is a reasonable first step to take to form the foundation of subsequent transcriptomic profiles from individual single-cells. Briefly, the overall research design is as follows: 1) standardize and optimize volume of cell sorting buffers for 10-, 50- and 100 cells; 2) use two types of external control RNA spike-in to correct for diverse total RNA in rare precursor cells; 3) guided by data from #2 to adjust for cell numbers to allow data normalization for between group comparisons with cell types with diverse RNA amount; 4) move forward with single-cell RNA-seq analysis guided by the most robust approaches available at that time (e.g., Fluidigm C1 or 10X Genomics and their single-cell RNA-seq kit)

项目概要/摘要项目负责人是 OSUCCC 基因组共享资源 (GSR) 和研究中心的技术总监俄勒冈州立大学医学院血液学系助理教授。她设立并指导了 Illumina测序运营至今已近9年。由于 GSR 服务于临床/转化项目 PI 获得了 OSU 的研究界和基础科学界的广泛见解与质量的影响有关（例如，物种、完整性、污染物的存在、保存过程、核酸提取方案）和输入材料的数量（浓度和总量）测序文库特征、测序数据质量和基因组中的读数分布。这份财富知识有助于开辟研究兴趣的另一个前沿（除了 DNA 甲基化和癌症表观遗传学）为 PI 生成 Biosketch 中突出显示的技术相关出版物列表文件以及正在准备的技术手稿，如研究策略 #3 和研究策略#5。对于 R50 应用程序，导师 PI（Muthusamy 博士和 Byrd 博士）均共享与在罕见病中正确控制和精心设计的低细胞数和单细胞 RNA-seq 相同的愿景 CLL 和 AML 中的前体细胞群将使我们能够评估 CLL 和 AML 中的白血病干细胞潜力 AML，检查这些稀有细胞群的药物敏感性和生物学机制。如果成功的话，这研究方向将为新疗法、复发预防和最终细胞水平开辟途径个性化医疗。由于人们对这些前体种群知之甚少，因此仔细描述它们的特征批量转录组图谱是形成后续转录组学基础的合理的第一步来自单个单细胞的概况。简而言之，总体研究设计如下：1）标准化和优化 10、50 和 100 个细胞的细胞分选缓冲液的体积； 2) 使用两种类型的外部对照RNA 添加以校正稀有前体细胞中不同的总 RNA； 3) 以 #2 的数据为指导来调整单元格数字允许数据标准化，以便与具有不同 RNA 量的细胞类型进行组间比较； 4) 在当时最强大的方法的指导下推进单细胞 RNA-seq 分析（例如 Fluidigm C1 或 10X Genomics 及其单细胞 RNA-seq 试剂盒）