Protoplasts were isolated from G.hirsutum cv. YZ1 embryogenic calli (described by Yang et al., 2008 (link)) and transformed with 35S::GhMYB4:MYC constructs. ChIP-based transient expression testing was performed as previously described with minor modification (Lee et al., 2017 (link)). Briefly, protoplasts were fixed with 1% (v/v) formaldehyde for 10 min at room temperature first, and the remaining formaldehyde was neutralized with 0.125-M glycine for 5 min. Fixed protoplasts were collected by centrifuging at 1,000g at 4°C for 5 min. Bioruptor was used at high power with 30-s-on/30-s-off cycles for 15 times until the average chromatin size was approximately 300 bp. Anti-MYC (Abcam, ab16898) antibodies were used to perform immunoprecipitations. Immune complexes were eluted from the protein A or G agarose/salmon sperm DNA beads (Millipore) and reverse cross-linked by incubation for 6 h at 65°C. Then qPCR reactions were performed. The primers are listed in Supplemental Table S5.