Chromatin Immunoprecipitation of SPL Transcription Factors

Eight-day-old transgenic seedlings of 35S::SPL9-HA or 35S::SPL15-HA were cross-linked with 1% formaldehyde and ground in liquid nitrogen. The chromatin complex was prepared following the method of Xie et al.^{24 (link)}. In brief, the supernatant was pre-cleared with 40 μl Protein-A-Agarose (16-157, EMD Millipore Corp.) and incubated at 4 °C for 1 h. Then the supernatant was moved into a microtube and 5 μl HA tag monoclonal antibodies (CB100005M, Cali-Bio) were added. After incubation at 4 °C for overnight with gentle agitation, 40 μl Protein-A-Agarose was added and incubated for 2 h. After washing, the immuno complex was eluted from the agarose beads. The precipitated DNA was then recovered and quantified using quantitative PCR with their individual primer pairs. The values were standardized to the input DNA to obtain the enrichment fold. PP2A was used as the internal control.