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Detection of DNA methylome in single-base resolution is a significant challenge but promises to shed considerable light on human disease etiology.In this study, we have developed MB-seq, a novel DNA methylome profiling technology combining MeDIP-seq with bisulfite conversion, which can precisely detect the 5mC sites and determine the differentially DNA methylation regions (DMRs) between DNA samples.Comparing MB-seq to whole-genome BS-seq, reduced representation bisulfite sequencing (RRBS), and MeDIP-seq using the same batch of DNA from T29 cell lines, we demonstrated that the method are powerful and cost-effective for genome-wide profiling of DNA methylation with high accuracy and sensitivity.In addition, we use MB to discriminate the first single base resolution DNA methylome of rat, and to detect tissue specific DMRs across the cerebral cortex, cerebellum and hypothalamus of rat.Taken together, our novel methods can accelerate the decoding process of DNA methylation mechanism in development and pathogenesis.