GSM5049269: MethylC-Capture Sequencing (MCC-seq) 6; Homo sapiens; OTHER
Semen samples were collected by masturbation after at least 3 days of abstinence. Following semen liquefaction (20-30min at room temperature), an aliquot was used for sperm count and the remaining sample was immediately frozen. ChIP-seq libraries were prepared using the Kapa HTP Library Preparation Kit (Roche) as per the manufacturer's recommendations except that the size selection was performed after the PCR step. For MCC-seq, bisulfite DNA was quantified with OliGreen® (Life Technology) and amplified with 9–12 PCR cycles using the KAPA® HiFi HotStart Uracil+ DNA Polymerase kit. SeqCap® Epi Enrichment System protocol (Roche NimbleGen®) was used to capture the regions of interest. Equal amounts of multiplexed libraries were combined to obtain 1μg of total input library, which was hybridized to the capture panel at 47°C for 72 h. Washing, recovery, and PCR amplification of the captured libraries, as well as final purification were conducted as recommended by the manufacturer (Roche/KAPA® Biosystems) according to suggested protocols.