Nano-MeDIP-seq: Methylome analysis using low DNA concentrations (Prot 58)
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 to minimise the drain on precious samples. MeDIP-seq fulfils these criteria, combining methylated DNA immunoprecipitation (MeDIP) with massively-parallel DNA sequencing. Methylated DNA Immunoprecipitation (MeDIP) is a technology capable of targeting the vast majority of the methylome. It involves antibodies directed against mC/mCG to precipitate methylated DNA fragments. MeDIP is able to detect methylated cytosines in both mC and mCG contexts. Because antibodies used for MeDIP were raised in a way to yield equal specificity against mC and mCG, MeDIP offers a near-unbiased and hypothesis-free approach without a priori assumptions about which regions of the methylome might be targeted (see below). Combining MeDIP with next generation sequencing (MeDIP-seq; Down et al., 2008), provides high-quality methylomes at typically 100-300bp resolution (depending on chosen insert size) at costs comparable to other capture-based techniques (Beck, 2010). In this EpiGeneSys Protocol Collection, which is based in an original publication in Nature Protocols (Taiwo et al., 2012), we detail Nano-MeDIP-seq – a protocol that uses 100-fold less genomic DNA than that which is commonly used for immunoprecipitation-based applications. Applications of this method will result in specific and sensitive enrichment of methylated DNA fragments over a wide range of DNA concentrations (5,000-50 ng), making Nano-MeDIP-seq suitable for studies involving minute clinical samples, micro-dissected tissues and rare cell types.
1 UCL Cancer Institute, University College London, London WC1E 6BT, UK.
2 UCL Institute of Healthy Ageing, University College London, London WC1E 6BT, UK
3 Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB22 3AT, UK
4 Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK
Corresponding author: Oluwatosin Taiwo, Stephan Beck
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