ChIP-qPCR Analysis of Peanut TFs

For the ChIP assay, leaves of four-leaf-stage (10–12 days after planting) peanuts (500 mg) were fixed with cold MC buffer (10 mM potassium phosphate pH 7.0, 50 mM NaCl, 0.1 M sucrose, 1% formaldehyde) for 20 min by vacuum concentration. Nucleoproteins were isolated by the method published by Su42 (link) and sonicated to produce DNA fragments on the order of 300 bp. Five μg anti-AhNAC2, anti-AhAREB1 and rabbit IgG (Millipore) were used for immunoprecipitation and antibody complexes were recovered by Protein G PLUS-Agarose (Santa Cruz Biotechnology). Specifically precipitated DNA was recovered and analyzed by real-time PCR with SYBR Premix ExTaq Mix (Takara Bio). The peanut genes ACTIN and AHD3 (Genbank: DQ873525.1 and EE127230.1, respectively) were used to calculate the relative fold-enrichment of target DNA fragments. The primers used to measure the binding of the TFs to the AhNCED1 promoter are listed in Supplemental Table 1.