Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: 18 DAP embryo chromatin was isolated as previously described (Yang et a;.2016).Briefly, formaldehyde-fixed samples were ground to fine powder in liquid nitrogen. Nuclei pellets were suspended in a buffer containing 0.25 M sucrose, 10 mM Tris-HCl, pH 8, 10 mM MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and protease inhibitors (one mini tablet per milliliter; Roche). The suspensions were transferred to microfuge tubes and centrifuged at 12,000g for 10 min. The pellets were suspended in 1.7 M sucrose, 10 mM Tris-HCl, pH 8, 2 mM MgCl2, 0.15% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and protease inhibitors (one tablet in 30 mL solution; Roche), and centrifuged through a layer of the same buffer in microfuge tubes. The nuclear pellets were lysed in a buffer containing 50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, and protease inhibitors (one tablet in 30 mL solution; Roche). The lysed nuclei were sonicated with a Bioruptor (UCD-200) in a water bath at 4°C for 12 cycles with 30s on and 30s off.Immunoprecipitation was performed using about 10 µg of chromatin, following a previously described protocol (Locatelli et al., 2009). Typically, 10 μL of affinity-purified antibody were used for immunoprecipitation. The precipitated DNA was dissolved in 50 μL 10 mM Tris-HCl, pH 7.5, 1 mM EDTA and treated with RNase (DNase-free). Antibody pulled-down DNA was subject to regular DNA library preparation procedure with NEBNext Ultra II DNA library Prep Kit (New England BioLabs).