dc.contributor.author | Meissner, Alexander | |
dc.contributor.author | Gnirke, Andreas | |
dc.contributor.author | Bell, George W. | |
dc.contributor.author | Ramsahoye, Bernard | |
dc.contributor.author | Jaenisch, Rudolf | |
dc.contributor.author | Lander, Eric Steven | |
dc.date.accessioned | 2012-09-17T18:26:08Z | |
dc.date.available | 2012-09-17T18:26:08Z | |
dc.date.issued | 2005-09 | |
dc.date.submitted | 2005-09 | |
dc.identifier.issn | 0305-1048 | |
dc.identifier.issn | 1362-4962 | |
dc.identifier.uri | http://hdl.handle.net/1721.1/73016 | |
dc.description.abstract | We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. BglII restriction fragments were size-selected to 500–600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We constructed RRBS libraries from murine ES cells and from ES cells lacking DNA methyltransferases Dnmt3a and 3b and with knocked-down (kd) levels of Dnmt1 (Dnmt[1[superscript kd],3a−/−,3b−/−]). Sequencing of 960 RRBS clones from Dnmt[1[superscript kd],3a−/−,3b−/−] cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. Non-CpG methylation was >250-fold reduced compared with wild-type ES cells, consistent with a role for Dnmt3a and/or Dnmt3b in CpA and CpT methylation. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt[1[superscript kd],3a−/−,3b−/−] cells. Near-complete bisulfite conversion and largely unbiased representation of RRBS libraries suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach. | en_US |
dc.description.sponsorship | National Institutes of Health (U.S.) (NIH Grant 5R01CA87869) | en_US |
dc.description.sponsorship | National Institutes of Health (U.S.) (NIH Grant HG03067-02) | en_US |
dc.description.sponsorship | Boehringer Ingelheim Fonds | en_US |
dc.language.iso | en_US | |
dc.publisher | Oxford University Press | en_US |
dc.relation.isversionof | http://dx.doi.org/10.1093/nar/gki901 | en_US |
dc.rights | Creative Commons Attribution Non-Commercial | en_US |
dc.rights.uri | http://creativecommons.org/licenses/by-nc/2.5 | en_US |
dc.source | Oxford | en_US |
dc.title | Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis | en_US |
dc.type | Article | en_US |
dc.identifier.citation | Meissner, A. “Reduced Representation Bisulfite Sequencing for Comparative High-resolution DNA Methylation Analysis.” Nucleic Acids Research 33.18 (2005): 5868–5877. Web. | en_US |
dc.contributor.department | Broad Institute of MIT and Harvard | en_US |
dc.contributor.department | Massachusetts Institute of Technology. Department of Biology | en_US |
dc.contributor.department | Whitehead Institute for Biomedical Research | en_US |
dc.contributor.approver | Jaenisch, Rudolf | |
dc.contributor.mitauthor | Lander, Eric S. | |
dc.contributor.mitauthor | Jaenisch, Rudolf | |
dc.contributor.mitauthor | Meissner, Alexander | |
dc.contributor.mitauthor | Gnirke, Andreas | |
dc.contributor.mitauthor | Bell, George W. | |
dc.relation.journal | Nucleic Acids Research | en_US |
dc.eprint.version | Final published version | en_US |
dc.type.uri | http://purl.org/eprint/type/JournalArticle | en_US |
eprint.status | http://purl.org/eprint/status/PeerReviewed | en_US |
dspace.orderedauthors | Meissner, A. | en |
mit.license | PUBLISHER_CC | en_US |
mit.metadata.status | Complete | |