ChIP-seq assay

ChIP-seq assays were performed on 14 day-old in vitro shoot seedlings using anti-H3K27me3 (Millipore 07–449) or anti- H3K27me1 (Millipore 07–448), following a procedure modified from Gendrel et al., 2005. Five grams of plantlets were cross-linked in 1% (v/v) formaldehyde at room temperature for 15min. Crosslinking was then quenched with 0.125 M glycine for 5 min. The crosslinked plantlets were ground and nuclei were isolated and lysed in Nuclei Lysis Buffer (1% SDS, 50 mM Tris-HCl pH 8, 10 mM EDTA pH 8). Cross-linked chromatin was sonicated using a water bath Bioruptor UCD-200 (Diagenode, Liège, Belgium) (15 s on/15 s off pulses; 15 times). The complexes were immunoprecipitated with antibodies, overnight at 4°C with gentle shaking, and incubated for 1 hr at 4°C with 40 µL of Protein AG UltraLink Resin (Thermo Scientific). The beads were washed 2 × 5 min in ChIP Wash Buffer 1 (0.1% SDS, 1% Triton X-100, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 150 mM NaCl),2 × 5 min in ChIP Wash Buffer 2 (0.1% SDS, 1% Triton X-100, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 500 mM NaCl), 2 × 5 min in ChIP Wash Buffer 3 (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate,10 mM Tris-HCl pH 8, 1 mM EDTA pH 8) and twice in TE (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8).ChIPed DNA was eluted by two 15 min incubations at 65°C with 250 μL Elution Buffer (1% SDS, 0.1 M NaHCO₃). Chromatin was reverse-crosslinked by adding 20 μL of NaCl 5M and incubated over-night at 65°C. Reverse-cross-linked DNA was submitted to RNase and proteinase K digestion, and extracted with phenol-chloroform. DNA was ethanol precipitated in the presence of 20 μg of glycogen and resuspended in 50 μL of nuclease-free water (Ambion) in a DNA low-bind tube. 10 ng of IP or input DNA was used for ChIP-Seq library construction using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer’s recommendations. For all libraries, 12 cycles of PCR were used. The quality of the libraries was assessed with Agilent 2100 Bioanalyzer (Agilent).