Reduced representation bisulfite sequencing (RRBS) is a method for large-scale, high-resolution analysis of DNA methylation patterns. The approach involves digesting genomic DNA with BglII, size-selecting fragments, adding adapters, bisulfite treatment, PCR amplification, cloning, and sequencing. This method was used to analyze DNA methylation in murine embryonic stem (ES) cells with and without functional DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b. RRBS libraries were constructed from these cells, and sequencing revealed that 99.9% of cytosines were converted to uracil, indicating high bisulfite conversion efficiency. Methylation levels were significantly reduced in Dnmt-deficient cells, with non-CpG methylation being 250-fold lower than in wild-type ES cells. The data suggest that methylation loss in these cells is random rather than specific. RRBS libraries provided high-quality, unbiased data suitable for genome-wide methylation studies. The method allows for efficient and accurate analysis of DNA methylation patterns across the genome, making it a valuable tool for epigenetic research. The study highlights the importance of DNA methyltransferases in maintaining methylation patterns and demonstrates the utility of RRBS for large-scale comparative epigenetic studies.Reduced representation bisulfite sequencing (RRBS) is a method for large-scale, high-resolution analysis of DNA methylation patterns. The approach involves digesting genomic DNA with BglII, size-selecting fragments, adding adapters, bisulfite treatment, PCR amplification, cloning, and sequencing. This method was used to analyze DNA methylation in murine embryonic stem (ES) cells with and without functional DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b. RRBS libraries were constructed from these cells, and sequencing revealed that 99.9% of cytosines were converted to uracil, indicating high bisulfite conversion efficiency. Methylation levels were significantly reduced in Dnmt-deficient cells, with non-CpG methylation being 250-fold lower than in wild-type ES cells. The data suggest that methylation loss in these cells is random rather than specific. RRBS libraries provided high-quality, unbiased data suitable for genome-wide methylation studies. The method allows for efficient and accurate analysis of DNA methylation patterns across the genome, making it a valuable tool for epigenetic research. The study highlights the importance of DNA methyltransferases in maintaining methylation patterns and demonstrates the utility of RRBS for large-scale comparative epigenetic studies.