GSM5379974: WT LFD sperm H3K4me3 rep 2; Mus musculus; ChIP-Seq
For sperm isolation, spermatozoa were collected from paired caudal epididymides as previously described (Hisano et al., 2013). ChIP-sequencing was performed using a previously described protocol (Hisano et al., 2013), 10 million spermatozoa used per sample. Samples were thawed on ice and washed with PBS. The chromatin was decondensed with 1M dithiothreitol and the reaction quenched with N-ethylmaleimide. Cells were lysed and aliquoted into 5 tubes and 15 units of MNase enzyme were added in each tube for 5 minutes, and the reaction was stopped with EDTA. Samples were centrifuged and the supernatants of aliquots from each sample were pooled back together. Pre-clearing of the chromatin was done for 1 hour and immunoprecipitation of the chromatin was performed for 8 hours with 5 µg of antibody targeting histone H3 lysine 4 trimethylation (H3K4me3). Samples were subjected to a 3-step wash, and the chromatin eluted and then treated with RNase A followed by Proteinase K. DNA was extracted and purified using the ChIP DNA Clean and Concentrator kit (Zymo Research) using the manufacturer's protocol. Mononucleosomes were selected size-selected before library preparation. Libraries were prepared using Qiagen's Ultra-low Input Library kit following manufacturer's protocol.