title: ChIP on 10-day old wheat seedlings: H3K9ac
design: Wheat seedlings were grown for 10 days in vitro at 18 °C in long-day conditions (16h light, 8h dark). After fixation with 1% (v/v) formaldehyde, tissues were homogenized, nuclei isolated and lysed. Cross-linked chromatin was sonicated using Covaris S220. Protein/DNA complexes were immunoprecipitated with antibodies overnight at 4°C with gentle shaking and incubated for 1h at 4°C with 50 ?L of Dynabeads Protein A (Invitrogen, Ref. 100-02D). The beads were washed 2 × 5 min in ChIP Wash Buffer 1 (0.1% SDS, 1% Triton X-100, 20mMTris-HCl pH 8, 2 mM EDTA pH 8, 150 mMNaCl), 2 × 5 min in ChIP Wash Buffer 2 (0.1% SDS, 1% Triton X-100, 20 mMTris-HCl pH 8, 2 mM EDTA pH 8, 500 mMNaCl), 2 × 5 min in ChIP Wash Buffer 3 (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 10 mMTris-HCl pH 8,1 mM EDTA pH 8) and twice in TE (10 mMTris-HCl pH 8, 1 mM EDTA pH 8). ChIPed DNA was eluted by two 15-min incubations at 65°C with 250?L Elution Buffer (1% SDS, 0.1 M NaHCO3). Chromatin was reverse-crosslinked by adding 20?L of NaCl 5M and incubating overnight at 65°C. Reverse-cross-linked DNA was treated with RNase and proteinase K, and extracted with phenol-chloroform. DNA was precipitated with ethanol in the presence of 20?g of glycogen and resuspended in 20?L of nuclease-free water (Ambion) in a DNA low-bind tube. 10 ng of IP or input DNA were used for ChIP-Seq library construction using NEB-Next Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's recommendations. For all libraries, ten cycles of PCR were used. The quality of the libraries was assessed with Agilent 2100 Bioanalyzer (Agilent), and the libraries were sequenced using 2x75bp pair-end reads on NextSeq 500 platform (Illumina).