Abstract
Development of high-throughput sequencing technologies now enables genome-wide analysis of DNA methylation of mammalian cells and tissues. Here, we present a protocol for Reduced Representation Bisulfite Sequencing (RRBS) applicable to low amounts of starting material (from 200 to 5,000 cells). RRBS is a cost-effective and powerful technique offering the advantages of absolute DNA methylation quantification and single nucleotide resolution while covering mainly CpG islands. Typically one sequencing experiment using the Illumina Genome Analyser IIx platform provides information on the DNA methylation status of more than half of the CpG islands of the mouse genome.
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Smallwood SA, Kelsey G (2012) De novo DNA methylation: a germ cell perspective. Trends Genet 28:33–42. doi:10.1016/j.tig.2011.09.004
Feng S, Jacobsen SE, Reik W (2010) Epigenetic reprogramming in plant and animal development. Science 330:622–627
Ferguson-Smith AC (2011) Genomic imprinting: the emergence of an epigenetic paradigm. Nat Rev Genet 12:565–575
Harris RA, Wang T, Coarfa C et al (2010) Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications. Nature Biotechnol 28:1097–1105
Bock C, Tomazou EM, Brinkman AB et al (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies. Nat Biotechnol 28:1106–1114
Ficz G, Branco MR, Seisenberger S et al (2011) Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473:398–402
Serre D, Lee BH, Ting AH (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
Lister R, Pelizzola M, Dowen RH et al (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462:315–322
Meissner A, Mikkelsen TS, Gu H et al (2008) Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454:766–770
Gu H, Bock C, Mikkelsen TS et al (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution. Nat Methods 7:133–136
Smallwood SA, Tomizawa S-I, Krueger F et al (2011) Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat Genet 43:811–814
Gu H, Smith ZD, Bock C et al (2011) Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nat Protoc 6:468–481
Illingworth RS, Gruenewald-Schneider U, Webb S et al (2010) Orphan CpG Islands identify numerous conserved promoters in the mammalian genome. PLoS Genet 6:e1001134
Quail MA, Kozarewa I, Smith F et al (2008) PERSPECTIVE A large genome center’s improvements to the Illumina sequencing system. Nat Methods 5:1005–1010
Krueger F, Andrews SR, Osborne CS (2011) Large scale loss of data in low-diversity illumina sequencing libraries can be recovered by deferred cluster calling. PLoS One 6:e16607
Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27:1571–1572
Acknowledgments
This work was supported by the Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Centre for Trophoblast Research.
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Smallwood, S.A., Kelsey, G. (2012). Genome-Wide Analysis of DNA Methylation in Low Cell Numbers by Reduced Representation Bisulfite Sequencing. In: Engel, N. (eds) Genomic Imprinting. Methods in Molecular Biology, vol 925. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-011-3_12
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DOI: https://doi.org/10.1007/978-1-62703-011-3_12
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Publisher Name: Humana Press, Totowa, NJ
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