Mouse sperm were isolated from 6 randomly selected, non-sibling males per generation. Epididymis and vas deferens were dissected and placed on a petri dish containing 2 mL of nucleus isolation medium (NIM) buffer (123.0 mmol/l KCl, 2.6 mmol/l NaCl, 7.8 mmol/l NaH2PO4, 1.4 mmol/l KH2PO4 and 3 mmol/l EDTA (disodium salt)). Sperm was pushed out of the tissue and media was collected and transferred to two clean microcentrifuge tubes and kept on ice. Samples were centrifuged at 13,000 xg for 5 minutes at 4ºC. Supernatant was removed and cells were resuspended in 1 mL of fresh NIM buffer. Sperm were counted using a hemocytometer, and 50,000 sperm were suspended in 22.5 uL water containing 0.01% digitonin. The diluted sperm were immediately subjected to tagmentation by adding 25 uL Nextera tagment DNA buffer (2X reaction buffer from Illumina Nextera kit) and 2.5 uL Nextera tagment DNA enzyme (Tn5 transposon enzyme from Nextera kit) followed by incubation at 37 °C for 30 minutes. Tagmented sperm DNA was immediately purified using QIAGEN MinElute kit and subjected to PCR amplification of the tagmented DNA fragments for Illumina deep sequencing library construction as previously described. To avoid batch effects, we randomly selected one sperm specimen from each of the four treatment groups – namely, F3 DMSO, F3 TBT, F4 DMSO, and F4 TBT – and performed the ATAC-seq reaction for these four specimens as a batch on the same day. We processed six batches (total 24 sperm specimens, 6 specimens for each treatment group) as independently performed experiments. The ATAC-seq libraries were sequenced using Illumina NextSeq 500 sequencer