ChIP-seq of Flag-Foxd4l1.1 and HA-Smad2/3

ChIP assay was performed as in a previous study [25 (link)]. The mRNAs encoding Flag-Foxd4l1.1 and HA-Smad2/3 (1 ng/embryo) were injected at the one-cell stage. The injected embryos were harvested at stage 11 (100 to 125 embryos/sample), and crosslinking was performed in 1.85% formaldehyde solution (Sigma-Aldrich, St. Louis, MO, USA). RIPA buffer containing proteinase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA) was added to fixed embryos, followed by homogenization and sonication for 90 s with 2 short intervals every 30 s to produce 200 to 300 base pair long fragments (Omni Sonic Ruptor 400). The anti-Flag and anti-HA polyclonal antibody (SC-805, Santa Cruz Biotechnology, Dallas, Texas, USA) or normal mouse IgG (SC-2025, Santa Cruz Biotechnology, Dallas, Texas, USA) were used to immunoprecipitate chromatin. The precipitated chromatin was then heated overnight at 65 °C to reverse the crosslinks, and the DNA was purified for further use. The ChIP-PCR was then performed with immunoprecipitated chromatin using region-specific primers (shown in Figure 5A). The primers used are listed in Table 5.