Single Cell Long Read Sequencing of the Aging Mouse Brain

National Institute On Aging

Investigators

Abstract

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.