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

项目摘要/摘要 该项目是OSUCCC基因组共享资源(GSR)的技术总监和一家研究机构 俄亥俄州立大学医学院血液科助理教授。她设立并执导了 Illumina测序作业至今已近9年。因为GSR同时服务于临床/转化性 在俄亥俄州立大学的研究界和基础科学界，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。这笔财富 知识有助于开辟研究兴趣的另一条战线(除了DNA甲基化和癌症 表观遗传学)导致生物草图中突出显示的与技术相关的出版物的列表 文件以及正在编写的技术手稿，如研究战略3和 研究策略5.对于R50应用程序，导师PI(Muthusamy博士和Byrd博士)都共享 同样的视觉，控制得当，设计良好，低细胞数和单细胞RNA-seq在罕见 CLL和AML中的前体群体将使我们能够评估CLL和AML中的白血病干细胞潜力 AML，研究这些稀有细胞群体的药物敏感性和生物学机制。如果成功，这将是 研究方向将为新的治疗方法、复发预防以及最终细胞水平开辟道路 个性化医疗。由于对这些前体种群知之甚少，仔细描述它们的特征 批量转录图谱是合理的第一步，以形成后续转录的基础 单个单细胞的配置文件。简而言之，总体研究设计如下：1)规范和 优化10细胞、50细胞和100细胞的细胞分选缓冲液的体积；2)使用两种类型的外控RNA 插入以校正稀有前体细胞中不同的总RNA；3)根据来自#2的数据来调整以适应细胞 允许对具有不同RNA量的细胞类型进行组间比较的数据标准化； 4)在当时最可靠的方法指导下，推进单细胞rna-seq分析。 (例如，Fluidigm C1或10X基因组学及其单细胞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)