title: Profiling DNA Methylation and Hydroxymethylation at Retrotransposable Elements.
abstract: DNA methylation is a key epigenetic modification controlling the transcriptional activity of mammalian retrotransposable elements. Its oxidation to DNA hydroxymethylation has been linked to DNA demethylation and reactivation of retrotransposons. Here we describe in detail protocols for three methods to measure DNA methylation and hydroxymethylation at specific genomic targets: glucMS-qPCR, and two sequencing approaches (pyrosequencing and high-throughput sequencing) for analyzing bisulfite- and oxidative bisulfite-modified DNA. All three techniques provide absolute measurements of methylation and hydroxymethylation levels at single-base resolution. Differences between the methods are discussed, mainly with respect to throughput and target coverage. These constitute the core techniques that are used in our laboratory for accurately surveying the epigenetics of retrotransposable elements.
authors.affiliation: Blizard Institute, School of Medicine and Dentistry, QMUL, E1 2AT, London, UK.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: de la Rica
authors.affiliation: Blizard Institute, School of Medicine and Dentistry, QMUL, E1 2AT, London, UK.
authors.forename: Jatinder S
authors.initials: JS
authors.lastname: Stanley
authors.affiliation: Blizard Institute, School of Medicine and Dentistry, QMUL, E1 2AT, London, UK. m.branco@qmul.ac.uk.
authors.forename: Miguel R
authors.initials: MR
authors.lastname: Branco
mesh_terms: D000818:Animals; D019295:Computational Biology; D018899:CpG Islands; D016172:DNA Fingerprinting / Q000379:methods*; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction; D018626:Retroelements; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D018626:Retroelements
keywords: Bisulfite; DNA hydroxymethylation; DNA methylation; High-throughput sequencing; Methylation-sensitive restriction enzyme; Oxidative bisulfite; Pyrosequencing; Quantitative PCR