Neuronal mosaicism describes the extent of intercellular genotypic diversity within a single human brain. This somatic variability is driven by numerous mechanisms including errors in DNA replication acquired throughout development and by the activity of endogenous retrotransposons. The study of retrotransposition in neuronal mosaicism may prove crucial to understanding the true complexity of normal and aberrant brain function. Specifically, numerous lines of evidence suggest that retrotransposition specific aspects of neuronal mosaicism may contribute to the unresolved etiology of many neurologic and neuropsychiatric disorders. Here, we describe the SLAV-Seq method, a recent advancement in the field over previous approaches used to study the diversity of LINE-1 based neuronal mosaicism at the single-cell level. We describe in detail, methodology for the isolation of single cells from bulk tissue by FACS, the amplification of single-cell genomic DNA by multiple displacement amplification (MDA), the targeted enrichment of LINE-1 somatic events, and the sequencing of the LINE-1 enriched library. Finally, we discuss methods for the quantification and analysis of the neuronal mosaicism identified by SLAV-Seq and some of the current technical limitations.
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Azevedo FAC, Carvalho LRB, Grinberg LT et al (2009) Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain. J Comp Neurol 513:532–541CrossRefPubMedGoogle Scholar
Kurimoto K, Yabuta Y, Ohinata Y et al (2007) Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis. Nat Protoc 2:739–752CrossRefPubMedGoogle Scholar
Christopher Love J, Ronan JL, Grotenbreg GM et al (2006) A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat Biotechnol 24:703–707CrossRefPubMedGoogle Scholar
Choi JH, Ogunniyi AO, Du M et al (2010) Development and optimization of a process for automated recovery of single cells identified by microengraving. Biotechnol Prog 26:888–895CrossRefPubMedGoogle Scholar
Rettig JR, Folch A (2005) Large-scale single-cell trapping and imaging using microwell arrays. Anal Chem 77:5628–5634CrossRefPubMedGoogle Scholar
Dean FB, Nelson JR, Giesler TL et al (2001) Rapid amplification of plasmid and phage DNA using phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res 11:1095–1099CrossRefPubMedPubMedCentralGoogle Scholar
Blanco L, Bernad A, Lázaro JM et al (1989) Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem 264:8935–8940PubMedGoogle Scholar
Garmendia C, Bernad A, Esteban JA et al (1992) The bacteriophage phi 29 DNA polymerase, a proofreading enzyme. J Biol Chem 267:2594–2599PubMedGoogle Scholar
Ulahannan D, Kovac MB, Mulholland PJ et al (2013) Technical and implementation issues in using next-generation sequencing of cancers in clinical practice. Br J Cancer 109:827–835CrossRefPubMedPubMedCentralGoogle Scholar