DNA methylation profiles of mouse sperm in response to antibiotic-induced gut microbiota dysbiosis using whole-genome bisulfite sequencing
DNA methylation profiles of mouse sperm in response to antibiotic-induced gut microbiota dysbiosis using whole-genome bisulfite sequencing
At 5 weeks of age, specific pathogen free (SPF) male C57BL/6J inbred mice were transferred to a conventional colony facility. The male mice were divided into separate cages (one mouse/cage) and randomly assigned to two groups: (i) Control group, which received regular diet and sterilized water for six successive weeks; (ii) Treated group, which received regular diet and sterilized water supplemented with a cocktail of non-absorbable antibiotics (neomycin 2.5 mg/ml, bacitracin 2.5 mg/ml and pimaricin 1.25ug/ml) for six successive weeks. The water bottle and ABX cocktail were replaced freshly every 5 days. At the end of six weeks treatment, male mice from both groups were mated with treatment-naive 6 weeks old C57BL/6J inbred female mice. Subsequent to mating, male mice were sacrificed and purified epididymal sperm were isolated as described previously (Sharma et al., 2016) and stored at -80C for BS-Seq Frozen sperm samples stored in -80C were thawed in ice; 100ml of DNA extraction buffer (20_mM Tris Cl pH 8, 20_mM EDTA, 200 mM NaCl, 4% SDS) containing 80 mM DTT and 12 microl/ml Proteinase K was added to the sample; and then incubated at 56C on Eppendorf thermomixer until complete sperm lysis assured (~ 1h), shake occasionally during incubation to disperse the sample. After incubation, extraction of total sperm DNA was performed using DNeasy Blood & Tissue Kit (QIAGEN Hilden, Germany) optimized for purification of sperm genomic DNA. BS-Seq libraries were constructed according to the manufacturerâ€™s instructions using TruSeq DNA Methylation Library Prep Kit (Illumina, CA, USA).