DNA methylation analysis for mouse spermatogonia defficient in Pld6.

In the male germline of mammals, the expression of retrotransposons is restricted by DNA methylation, trimethylation of histone H3 at lysin-9 (H3K9me3) and PIWI-interacting small RNAs (piRNAs). To elucidate their relative importance in regulating retrotransposons during germ-cell development as well as relationships between these mechanisms, we performed mRNA, DNA methylation, and histone methylation analyses using mouse spermatogonia from Dnmt3l and Pld6 mutants deficient in de novo DNA methylation and piRNA production, respectively. The results revealed that a loss of DNA methylation results in decreased H3K9me3 and increased H3K4me3, suggesting a pivotal role of DNA methylation in maintaining the epigenome integrity. Overall design: DNA was extracted by phenol chloroform extraction from wild-type and Pld6 KO mouse spermatonia at postnatal day 7, which were purified by FACS (EPCAM+). DNA was then fragmented by sonication on Bioruptor for 15 cycles of high output for 30 seconds. After purification by AMPure, DNA was treated with high concentration of bisulfite at 70 degree for an hour, then purified using EZ methylation kit. BS-seq libraries were made using ACCEL-NGS methyl-seq library Kit (Swift Biosciences) and sequenced on HiSeq Ten X with a 150-bp paired-end mode.