GSM6284582: HFD sperm H3K4me3 rep 1; 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, Lismer et al., 2021). Sperm from 2-7 animals were pooled for a total of 12 million spermatozoa used per sample. Samples were thawed on ice and washed with PBS. Dithiothreitol (1M) was used to decondense the chromatin and N-ethylmaleimide  to quench the reaction. Cells were lysed and aliquoted into 6 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 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, 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. Libraries were prepared for illumina sequencing, and samples were sequenced on the illumina HiSeq2500 platform with 100 bp single-end reads.