library_construction_protocol: Male Xenopus tropicalis were injected with 150 U HCG. After 2 hours, two X. tropicalis testes collected in 1 ml 1xMBS and homogenized using razor blades. The solution was filtered with 40um strainer and its volume was brought down to 14ml. Then, through twice 180G horizontal centrifugation, we can obtain pure sperm in the supernatant. The ChIP assay was performed as described. Briefly, 5-20 millions sperm were crosslinked with 1% formaldehyde for 10 min using vacuum infiltration. (Mouse cells were added as spike-in control.) Chromatin was sheared to an average size of 150 bp using a sonicator (Bioruptor Pico, Diagenode). Sonicated chromatin fragments were immunoprecipitated with 3-5 μg of antibody. Chromatin-bound antibodies were recovered with 30μl Protein A/G magnetic beads (Millipore 16-663). After reverse crosslinking, ChIPed DNA was recovered using the MinElute Reaction Cleanup Kit (Qiagen 28206) and amplified with the VAHTS® Universal DNA Library Prep Kit for Illumina V3 (Vazyme ND607). Amplified ChIP libraries were sequenced on the Illumina HiSeq X10 platform.