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Staphylococcus aureus intramammary infection is one of the most common causes of chronic mastitis in dairy cows, whose development may be associated with epigenetic changes in the expression of important host defense genes. This study aimed to construct a genome-wide DNA methylation profile of the mammary gland of Chinese Holstein cows (n = 3) following experimentally induced S. aureus mastitis, and to explore the potential gene regulatory mechanisms affected by DNA methylation during S. aureus mastitis. DNA was extracted from S. aureus