Retrotransposon expression analysis for mouse spermatogonia defficient in Dnmt3l and 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: RNA was extracted from wild-type, Dnmt3l KO, and Pld6 KO mouse spermatonia at postnatal day 7, which were purified by FACS (EPCAM+). mRNA-seq libraries were made using NEBNext Ultra II Directional mRNA library prep kit for illumina (New England Biolab). Sequencing was performed on HiSeq2500 (Illumina) using 50-bp single-end mode.