2.5. ChIP assay

Ten MDs at E14.5 were sampled in 1% Protease Inhibitor Cooktail (Wako; added to all buffer by chromatin extraction)/PBS, and crosslinked with 1.5% PFA. Fixation was stopped by addition of glycine. The samples were washed with PBS and homogenized. The pellets were lysed in 0.5% NP40 buffer (10 mM Tris (pH8.0), 10 mM NaCl), and then lysed in 1% SDS buffer (50 mM Tris (pH8.0), 10 mM EDTA). The samples were added with ChIP dilution buffer (50 mM Tris (pH8.0), 167 mM NaCl, 1.1% Triton X-100 and 0.11% Sodium Deoxycholate). The chromatins were sheared with a sonicator to an average length of 200 bp.

Thirty μl ChIP-grade protein G magnetic beads (Cell Signaling Technologies, Beverly, MA, USA) and 0.5 μg anti-RNA polymerase II CTD repeat antibody (Santa Cruz biotechnology, Santa cruz, CA, USA), anti-CEBPδ antibody (Santa Cruz Biotechnology) or normal rabbit IgG (Santa Cruz Biotechnology) were mixed in RIPA buffer and incubated at 4°C overnight. After washing with RIPA buffer, the mixtures and the fragmented chromatins were mixed and incubated at 4°C overnight. Samples were then sequentially washed in RIPA buffer, RIPA buffer containing 500 mM NaCl, LiCl wash solution and TE buffer, and eluted in 0.5% SDS buffer (10 mM Tris (pH 8.0), 300 mM NaCl, 5 mM EDTA) at 65°C for 8 h, then added with protease K at 500 μg/ml and incubated for 1 h at 55°C. Proteins were removed by phenol and chloroform and DNAs were precipitated by ethanol. The DNAs were compared with inputs by quantitative PCR using KOD SYBR qPCR Mix (TOYOBO). Aldh1a2 TSS (−89 to +11), Aldh1a2 C/EBPδ predicted binding site (+2872 to +3987) and Aldh1a2 conserved region primer (−381 to +1, 25,794 to +26,539) were derived from Gene ID 19,378 (Supplemental Table).