Advancing Epigenetics Towards Systems Biology

Nano-MeDIP-seq: Methylome analysis using low DNA concentrations (Prot 58)

Oluwatosin Taiwo 1,2, Gareth A Wilsona, Tiffany Morris1, Stefanie Seisenberger3, Wolf Reik3,4, Daniel Pearce2, Stephan Beck1 & Lee M Butcher1

Introduction

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.

PDF version

Oluwatosin Taiwo 1,2, Gareth A Wilsona, Tiffany Morris1, Stefanie Seisenberger3, Wolf Reik3,4, Daniel Pearce2, Stephan Beck1 & Lee M Butcher1

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
Email feedback to: This email address is being protected from spambots. You need JavaScript enabled to view it., This email address is being protected from spambots. You need JavaScript enabled to view it.

20130221112824 p58