![]() 5-Methylcytosine is the only known covalent modification of DNA in vertebrates ( 1 ). 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.ĭNA methylation is a key epigenetic modification that provides heritable information not encoded in the nucleotide sequence. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt cells. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. 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. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Sequencing of 960 RRBS clones from Dnmt cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. 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 ). BglII restriction fragments were size-selected to 500–600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns.
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