title: Methylome analysis using MeDIP-seq with low DNA concentrations.
abstract: DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000-50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160-300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3-5 d.
authors.affiliation: University College London (UCL) Cancer Institute, University College London, London, UK. t.taiwo@ucl.ac.uk
authors.forename: Oluwatosin
authors.initials: O
authors.lastname: Taiwo
authors.forename: Gareth A
authors.initials: GA
authors.lastname: Wilson
authors.forename: Tiffany
authors.initials: T
authors.lastname: Morris
authors.forename: Stefanie
authors.initials: S
authors.lastname: Seisenberger
authors.forename: Wolf
authors.initials: W
authors.lastname: Reik
authors.forename: Daniel
authors.initials: D
authors.lastname: Pearce
authors.forename: Stephan
authors.initials: S
authors.lastname: Beck
authors.forename: Lee M
authors.initials: LM
authors.lastname: Butcher
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D053938:DNA Fragmentation; D019175:DNA Methylation; D057890:Epigenomics / Q000379:methods*; D005434:Flow Cytometry; D015723:Gene Library; D047468:Immunoprecipitation / Q000379:methods; D051379:Mice; D017422:Sequence Analysis, DNA / Q000379:methods