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dc.contributor.authorMeissner, Alexander
dc.contributor.authorGnirke, Andreas
dc.contributor.authorBell, George W.
dc.contributor.authorRamsahoye, Bernard
dc.contributor.authorLander, Eric S.
dc.contributor.authorJaenisch, Rudolf
dc.date.accessioned2012-09-17T18:26:08Z
dc.date.available2012-09-17T18:26:08Z
dc.date.issued2005-09
dc.date.submitted2005-09
dc.identifier.issn0305-1048
dc.identifier.issn1362-4962
dc.identifier.urihttp://hdl.handle.net/1721.1/73016
dc.description.abstractWe 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.sponsorshipNational Institutes of Health (U.S.) (NIH Grant 5R01CA87869)en_US
dc.description.sponsorshipNational Institutes of Health (U.S.) (NIH Grant HG03067-02)en_US
dc.description.sponsorshipBoehringer Ingelheim Fondsen_US
dc.language.isoen_US
dc.publisherOxford University Pressen_US
dc.relation.isversionofhttp://dx.doi.org/10.1093/nar/gki901en_US
dc.rightsCreative Commons Attribution Non-Commercialen_US
dc.rights.urihttp://creativecommons.org/licenses/by-nc/2.5en_US
dc.sourceOxforden_US
dc.titleReduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysisen_US
dc.typeArticleen_US
dc.identifier.citationMeissner, 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.departmentBroad Institute of MIT and Harvarden_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Biologyen_US
dc.contributor.departmentWhitehead Institute for Biomedical Researchen_US
dc.contributor.approverJaenisch, Rudolf
dc.contributor.mitauthorLander, Eric S.
dc.contributor.mitauthorJaenisch, Rudolf
dc.contributor.mitauthorMeissner, Alexander
dc.contributor.mitauthorGnirke, Andreas
dc.contributor.mitauthorBell, George W.
dc.relation.journalNucleic Acids Researchen_US
dc.eprint.versionFinal published versionen_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.orderedauthorsMeissner, A.en
mit.licensePUBLISHER_CCen_US


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