Abstract
The analysis of DNA-methylation on a genome-wide scale by next-generation sequencing techniques is an invaluable tool towards the understanding of the epigenetic basis of cellular differentiation. Methylated DNA immunoprecipitation (MeDIP) is an immunocapturing method using an antibody targeting 5-methylcytidine (5mC) and thereby enriching methylated DNA. MeDIP combined with next-generation sequencing (MeDIP-seq) provides a powerful tool for the analysis of genome-wide DNA-methylation profiles. Here, we describe a protocol for the preparation of MeDIP samples suitable for next-generation sequencing on a Genome Analyser (Illumina).
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Lister R, Pelizzola M, Dowen RH et al (2009) Human DNA methylomes at base resolutionshow widespread epigenomic differences. Nature 462:315–322
Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929–930
Song CX, Szulwach KE, Fu Y et al (2011) Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol 29:68–72
Meissner A, Mikkelsen TS, Gu H et al (2008) Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454:766–770
Harris RA, Wang T, Coarfa C et al (2010) Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications. Nat Biotechnol 28:1097–1105
Serre D, Lee BH, Ting AH et al (2010) MBD-isolated genome sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome. Nucleic Acids Res 38:391–399
Bock C, Tomazou EM, Brinkman AB et al (2010) Quantitative comparison of genome-wide DNAmethylation mapping technologies. Nat Biotechnol 28:1106–1114
Down TA, Rakyan VK, Turner DJ et al (2008) A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis. Nat Biotechnol 26:779–785
Ruike Y, Imanaka Y, Sato F et al (2010) Genome-wide analysis of aberrant methylation in human breast cancer cells using methyl-DNA immunoprecipitation combined with high-throughput sequencing. BMC Genomics 25(11):137
Maunakea AK, Nagarajan RP, Bilenky M et al (2010) Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466:253–257
Chavez L, Jozefczuk J, Grimm C et al (2010) Computational analysis of genome-wide DNA methylation during the differentiation of human embryonic stem cells along the endodermal lineage. Genome Res 20:1441–1450
Jin SG, Kadam S, Pfeifer GP (2010) Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res 38:e125
Weber M, Davies JJ, Wittig D et al (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DANN methylation in normal and transformed human cells. Nat Genet 37:853–862
Butcher LM, Beck S (2010) AutoMeDIP-seq: a high-throughput, whole genome, DNA methylation assay. Methods 52:223–231
Acknowledgments
CG and JA are funded by the German Ministry of Education and Research (BMBF) within its National Genome Research Network (01GS08111) and the ERASysBio Plus programme (0315717A). We thank Justyna Jozefczuk for providing genomic DNA of undifferentiated human embryonic stem cells and the corresponding endoderm-differentiated cells used for the qPCR assay in Fig. 3 and Aleksey Soldatov, Tatiana Borodina and Aleksey Kirpy for advice on library preparation. We thank Vardhman Rakyan and Stephan Beck for helpful discussions regarding the MeDIP-seq protocol and for providing the human control primer sequences prior to publication.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Grimm, C., Adjaye, J. (2012). Analysis of the Methylome of Human Embryonic Stem Cells Employing Methylated DNA Immunoprecipitation Coupled to Next-Generation Sequencing. In: Turksen, K. (eds) Human Embryonic Stem Cells Handbook. Methods in Molecular Biology, vol 873. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-794-1_19
Download citation
DOI: https://doi.org/10.1007/978-1-61779-794-1_19
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-793-4
Online ISBN: 978-1-61779-794-1
eBook Packages: Springer Protocols