At PND 90 the male rats for F1, F2 and F3 generations were anesthetized by isofurane and euthanized by cardiac puncture. Fresh right caudal epididymis was placed in a 35 mm Petri dish with prewarmed Gibco®Media-199 without phenol red (Life Technologies), 0.5% fatty acid-free BSA (Fraction V; Sigma-Aldrich,St.), nicked scalpel several times and then incubated for 10 min in a humidified 5% CO2 incubator at 37°C to allow sperm to diffuse. 500 ng of DNA (PureLink genomic DNA kit Invitrogen) were used for RRBS libraries (H. Gu et al. Nature protocols 6, 468, (2001)) using the gel-free technique. The bisulfite conversion and subsequent clean-up were done by using the EpiTect bisulfite kit (Qiagen). Libraries PCR was realized with PfuTurbo Cx hotstart DNA polymerase (Agilent Technologies).
GEO Accession
GSM3041226