Protein a or g agarose beads

For ChIP analysis with NIGT1.1-OX #9 plants, seedlings were grown for 10 days on agar plates that contained 1/2MS salts, 3 mM MES-KOH (pH 5.8), 0.5% sucrose and 0.8% agar. For ChIP analysis with PHR1-OX plants, etiolated seedlings were generated by cultivation for 1 day under continuous illumination and then 2 days in the dark. Plants were then hydroponically grown with 1/10MS solution for 13 days and then 1/10MS solution without P for 5 days. ChIP was performed using the protocol of Saleh et al.^{69 (link)} with slight modifications. In brief, after cross-linking proteins to DNA and isolation and lysis of nuclei, DNA sonication was performed with a BIORUPTOR^®II (COSMO BIO CO., LTD, Tokyo, Japan) at high mode, 10× 30 s, with 30-s intervals. Immunoprecipitation was carried out with Protein A or G-agarose beads (Roche) conjugated to 5 µg of anti-MYC antibody (clone 9E10, Millipore #05-419) overnight at 4 °C. DNA recovered from agarose beads was purified using a DNeasy Plant Mini Kit (Qiagen). qPCR was performed with a StepOne Plus™ Real Time PCR System (Applied Biosystems) and the KAPA SYBR Fast qPCR Kit (KAPA Biosystems). Three or four biological samples were analysed with consistent results.

For each Immunoprecipitation, 10 million cells were lysed in buffer I (0.5% NP-40, 10 mM Hepes, 10 mM KCl, 2 mM EDTA, 10% Glycerol, Complete protease inhibitor (Roche), pH7.5), incubated on ice for 10 min and centrifuged for 10 min at 13,000 rpm. Pellets were lysed in 500 μl buffer II (50 mM Sodium Phosphate, 300 mM NaCl, 1 mM β-mercaptoethanol, 10% Glycerol, 0.5% NP-40, 0.5% Triton X-100, Complete protease inhibitor (Roche), pH 7.5), mixed by vortexing and sonicated twice on a Brason digital sonifier for 10 sec at 50% output followed by 10 min incubation on ice and centrifuged for 10 min at 13,000 rpm. Supernatant was incubated for 2 h using the antibody of interest followed by 1 h incubation with Protein-A or -G agarose beads (Roche). Beads were washed 4 times with buffer II and proteins were extracted by boiling the beads for 5 min in SDS-PAGE sample loading buffer prior to separation by SDS-PAGE electrophoresis.