GSM8183032: Sperm, Chow Diet, Rep6; Mus musculus; ATAC-seq
Mouse mature sperm extraction was carried out in accordance with the protocol used for murine in vitro fertilization. For each dissection, a Petri dish was prepared containing 1mL of 1x PBS at its center, subsequently overlaid with paraffin oil. Following euthanasia of the male mouse by cervical dislocation, both caudae epididymides were excised and positioned on the paraffin oil in the Petri dish. Each cauda was incised midway using scissors, releasing the sperm clusters, which were then coaxed towards the 1x PBS at the dish's center. After a 10-minute incubation at room temperature, the PBS containing the sperm was collected and centrifuged at 600g at 4ºC for 5 minutes. The sperm was then washed twice with 1x PBS and subjected to counting. After mature sperm isolation ATAC-seq was performed following the Omni-ATAC protocol. Briefly, the purified sperm pellet was resuspended in the transposase reaction mix containing 1% of digitonin and incubated for 30min at 37ºC. Following incubation, sperm were treated with proteinase K at 55ºC for 2h and genomic DNA (gDNA) was isolated by Phenol:Chlorophorm:Isoamyl alcohol (PCI) and subsequent EtOH precipitation. Library amplification was performed using 2X KAPA HiFi mic and 25uM indexed primers, with PCR conditions as it follows: 72ºC for 5min; 98ºC for 30s; and 8-11 cycles at 98ºC for 10s; 63ºC for 30s; and 72ºC for 1min. Each ATAC-sew experiment was conducted with 4 and 6 biological replicates on Chow diet (F0 CD) and Western diet (F0 WD) groups respectively, and each one contained at least 60 million reads after all the quality control steps.