ChIP-qPCR Analysis of OsNAC42 Binding

The ChIP assay was adapted from Zhao et al.^{45 (link)}. Total DNA of three-week-old seedlings of Nipponbare rice was extracted. The total DNA was sheared into 100–500 bp fragments using an ultrasonic crusher. E.coli expressed MBP-OsNAC42 and MBP-OsNAC42M proteins were purified. MBP-OsNAC42, MBP-OsNAC42M and sheared rice DNA fragments were co-incubated with amylase Resin (NEB, E8021S, MBP beads) for 8 h. The incubation buffer included: 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 1% methanol. After 8 hours co-incubation, 125 mM glycine was further added into the beads mixture and incubated for 10 min. MBP beads were washed three times using incubation buffer. For each of 400 ml volume of the samples, add 16 μL 5 M NaCl and was incubated for another 8 h to breakdown associated DNA with MBP-OsNAC42 and MBP-OsNAC42M proteins. Subsequently, DNA fragments were extracted using the phenol–chloroform method for further ChIP-qPCR analysis with a Bio-Rad CFX96 real-time PCR detection system. PCR were performed in triplicate for each sample, and the expression levels were normalized to the input sample and only MBP control treatment for enrichment detection. The enrichment folds were calculated against the amount of bound DNA of rice ubiquitin promoter. Relevant primer sequences were given in Supplementary Data 2.