Single Cell Long Read Sequencing of the Aging Mouse Brain

衰老小鼠大脑的单细胞长读测序

• 批准号: 10688815
• 负责人: Emmanouil Maragkakis
• 金额: $ 95.43万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10688815
• 关键词: Aging; Alternative Splicing; Amino Acid Sequence Databases; Animals; Brain; Brain region; Cell Aging; Cells; Cerebral cortex; Chromium; Complementary DNA; Coupled; Data; Data Set; Evaluation; Event; Female; Genomics; Heterogeneity; Hippocampus (Brain); Intergenic Sequence; Knowledge; Length; Mass Spectrum Analysis; Methods; Mus; Nerve Degeneration; Peptides; Protein Isoforms; Proteins; Proteomics; System; Techniques; Transcript; Validation; Variant; Work; aging brain; base; male; nanopore; novel; novel marker; proteogenomics; single cell analysis; single cell sequencing; single-cell RNA sequencing; transcriptome; transcriptome sequencing; young adult

Single-cell RNA sequencing is a key technique for the analysis of cell-to-cell heterogeneity. While droplet-based high throughput scRNA-seq approaches, including the 10x Genomics Chromium controller system, allow the analysis of thousands of cells, they only yield limited sequence information biased towards the 3 end of the transcript. Therefore, information on the full-length transcriptome, including evaluation of alternate splicing, is not able to be derived from typical scRNA-Seq approaches. Here, we employ single-cell nanopore sequencing to skip the cDNA fragmentation step and sequence full-length cDNA molecules. We compare young adult and old mouse brains, including both male and female animals. Acquired data are compared to existent single cell datasets to discover cell-to-cell transcriptome heterogeneity in aging. RNA sequencing is coupled with mass spectrometry (MS)-based proteomics for the protein-level validation of transcript variants, isoforms, intra/inter-genic sequences, and other novel transcripts identified by RNA sequencing. To generate protein sequence databases, we convert RNA-seq data into refined protein sequence database containing novel proteins, reference proteins, and decoy proteins used to estimate FDRs for peptide identifications. Collectively, this work aims to identify new markers of the brain and substantially refine knowledge of brain aging.

单细胞RNA测序是分析细胞间异质性的关键技术。虽然基于液滴的高通量scRNA-seq方法，包括10 x Genomics Chromium控制器系统，允许分析数千个细胞，但它们仅产生偏向转录物3端的有限序列信息。因此，关于全长转录组的信息，包括选择性剪接的评估，不能从典型的scRNA-Seq方法中获得。在这里，我们采用单细胞纳米孔测序来跳过cDNA片段化步骤并对全长cDNA分子进行测序。我们比较了年轻的成年小鼠和老年小鼠的大脑，包括雄性和雌性动物。将获得的数据与现有的单细胞数据集进行比较，以发现衰老中的细胞间转录组异质性。RNA测序与基于质谱（MS）的蛋白质组学相结合，用于转录变体、同种型、基因内/基因间序列和通过RNA测序鉴定的其他新转录物的蛋白质水平验证。为了生成蛋白质序列数据库，我们将RNA-seq数据转换为包含新蛋白质、参考蛋白质和诱饵蛋白质的精细蛋白质序列数据库，用于估计肽鉴定的FDR。总的来说，这项工作的目的是确定大脑的新标记，并大大完善大脑衰老的知识。