Abstract
Enormous heterogeneity in transcription and signaling is the feature that slows down progress in our understanding of the mechanisms of normal aging and age-related diseases. This is critical for neurobiology of aging where the enormous diversity of neuronal populations presents a significant challenge in experimental design. Here, we introduce Aplysia as a model for genomic analysis of aging at the single-cell level and provide protocols for integrated transcriptome and methylome profiling of individually identified neurons during the aging process. These single-cell RNA-seq and DNA methylation assays (methyl-capture/methyl enrichment) are compatible with all major next generation sequencing platforms (we used Roche/454 and SOLiD/Life Technologies as illustrative examples) and can be used to integrate an epigenetic signature with transcriptional output. The described sequencing library construction protocol provides both quantitative and directional information from transcriptional profiling of individual cells. Our results also confirm that different copies of DNA in polyploid Aplysia neurons behave similarly with respect to their DNA methylation.
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Acknowledgements
We thank Mr. James Netherton for reading and commenting on the manuscript. We would like to thank Dr. Clarence Lee for help with the MethylMiner libraries. We also thank Dr. Manfred Lee for his technical advice and guidance with the Ion PGM sequencing process as well as anonymous reviewers for their critical comments and suggestions. We also thank Dr. Thomas Ha, Dr. Sami Jezzini, and Mrs. Yelena Bobkova for help with tissue preparations and RNA/DNA quality assays. This work is supported by NIH grants 1R01GM097502, R21RR025699, 5R21DA030118, R01MH097062, McKnight Brain Research Foundation, as well as NSF-0744649, NSF CNS-0821622, and UF Opportunity Fund awards to LLM.
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Moroz, L.L., Kohn, A.B. (2013). Single-Neuron Transcriptome and Methylome Sequencing for Epigenomic Analysis of Aging. In: Tollefsbol, T. (eds) Biological Aging. Methods in Molecular Biology, vol 1048. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-556-9_21
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