library_construction_protocol: For Adnp knockout, three single guide RNAs (sgRNAs) targeting the last exon were designed. To assemble Cas9–sgRNA ribonucleoproteins, 200 ng μl−1 sgRNAs was mixed with 0.3 μl of Alt-R S.p. HiFi Cas9 protein (Integrated DNA Technologies, 1081060) and incubated at room temperature for 15 min in 15 μl assembly buffer (1 mM Tris-HCl pH 7.5, 0.1 mM EDTA). The injection mix was centrifuged at 12,000g for 10 min, and the supernatant was used for injection into PN3 zygotes. Embryos were then cultured in G1 medium (Vitrolife). Blastocyst-stage embryos were collected for RNA-seq, CUT&RUN and Hi-C. For DRB treatment, 8C embryos (68–70 h after hCG injection) were collected and cultured in CZB medium supplemented with DRB (final concentration 120 μM) for 2 or 4 h.For the cohesin-KD experiment, PN3 zygotes were injected with 20 μM siRNAs (targeting Rad21 (Rad21 KD) or Nipbl, Smc3 and Rad21 (triple KD; Supplementary Table 2). The PN3 zygotes in the control group were injected with an equal volume of 20 μM scramble siRNAs. Then the embryos were cultured in G1 medium (Vitrolife) for 45 h. Embryos were crosslinked with 1% formaldehyde at room temperature for 10 min and then quenched by glycine for 10 min at room temperature. Embryos were then washed by PBS and lysed on ice and solubilized with 0.5% SDS. The nuclei were digested with MboI at 37 °C for 12 h. The resulting fragments were filled with biotin-14-dATP and then ligated. After ligation, crosslink reversal, DNA purification and sonication were performed sequentially, and the biotin-14-dATP-labelled targets were pulled down by Dynabeads MyOne Streptavidin C1 (Life Technology). The purified DNA was subjected to the True-seq library construction process.