Flow Cytometric and Sorting Analyses for Nuclear DNA Content, Nucleotide Sequencing, and Interphase FISH
The study of genomic mosaicism among human brain cells is challenging. The human brain contains hundreds of billions of cells that are intricately connected and difficult to separate as intact, single cells. Additional challenges are encountered when interrogating small, seemingly random changes within single-cell genomes. Flow cytometric analysis (FCM), and fluorescence-activated nuclear sorting (FANS), has expanded our assessment capabilities for global and specific genomic and transcriptomic changes in human brain cells. The general approach is being utilized in a variety of downstream applications by many laboratories. Here we provide detailed methods of nuclear DNA content assessment and sorting that reports population averages as well as single-cell nuclear DNA content from cells of the human brain. We highlight protocol modifications that allow the same nuclear preparation to be used for subpopulation-specific FANS (also see chapter “Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity”) in downstream analyses such as fluorescent in situ hybridization (FISH) (see chapters “FISH-Based Assays for Detecting Genomic (Chromosomal) Mosaicism in Human Brain Cells,” “FISH Analysis of Aging-Associated Aneuploidy in Neurons and Non-neuronal Brain Cells” and “Using Fluorescence In Situ Hybridization (FISH) Analysis to Measure Chromosome Instability and Mosaic Aneuploidy in Neurodegenerative Diseases”), and single-cell genomic and transcriptomic sequencing (see chapters “Flow Cytometric Quantification, Isolation, and Subsequent Epigenetic Analysis of Tetraploid Neurons,” “Single Cell CNV Detection in Human Neuronal Nuclei,” “Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) for the Analysis of DNA Copy Number Variation,” and “Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity”). Other downstream techniques include, but are not limited to, single-cell qPCR (see chapter “Competitive PCR for Copy Number Assessment by Restricting dNTPs”) and estimation of line-1 copy number (see chapters “Analysis of LINE-1 Retrotransposition in Neural Progenitor Cells and Neurons,” “Estimation of LINE-1 Copy Number in the Brain Tissue and Isolated Neuronal Nuclei,” and “Analysis of Somatic LINE-1 Insertions in Neurons”).
Key wordsDNA content variation Flow sorting Flow cytometry Neuron Nuclei NeuN Sequencing Fish Genomic mosaicism Somatic Aneuploidy Aneusomy
The authors are funded by the NIH Common Fund Single Cell Analysis Program (1U01MH098977), the NIAAA (4R01AA021402), and the Neuroplasticity of Aging Training Grant (5T32AG000216). We thank Danielle Jones, Dr. Ming Hsiang Lee, and Suzanne Rohrback for their contributions to this manuscript.
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