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

项目总结/摘要 项目PI是OSUCCC基因组学共享资源（GSR）的技术总监， 俄勒冈州立大学医学院血液学系助理教授。她建立并指导了 Illumina测序操作至今已近9年。由于GSR既服务于临床/翻译， 在俄勒冈州立大学的研究社区和基础科学社区，PI项目获得了广泛的见解 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段分布。这些丰富的 知识有助于开辟另一个研究领域（除了DNA甲基化和癌症之外 表观遗传学），导致在Biosketch中突出显示技术相关出版物列表 文件以及研究策略#3中描述的正在编写的技术手稿， 研究策略#5对于R50应用程序，Mentor PI（Muthusamy博士和Byrd博士）共享 同样的愿景，适当控制和精心设计的低细胞数和单细胞RNA-seq在罕见的 CLL和AML中的前体细胞群将使我们能够评估CLL中白血病干细胞的潜力， AML，研究这些罕见细胞群的药物敏感性和生物学机制。如果成功，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。 个性化医疗由于对这些前体种群知之甚少， 批量转录组学谱是形成后续转录组学谱的基础的合理的第一步。 单个细胞的特征。简要地说，总体研究设计如下：1）标准化， 优化用于10个、50个和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 加标以校正稀有前体细胞中的不同总RNA; 3）由来自#2的数据指导以调整细胞 允许数据标准化的数字，用于与具有不同RNA量的细胞类型进行组间比较; 4)在当时最强大的方法指导下，继续进行单细胞RNA-seq分析 (e.g., Fluidigm C1或10 X Genomics及其单细胞RNA-seq试剂盒）

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)