alias: RcHt_H3K27me3_mark_Petal
title: H3K27me3 ChipSeq data from Rose petal
design: ChIP assays were performed using anti-H3K9ac (Millipore, ref. 07-352) or anti-H3K27me3 (Millipore, ref. 07-449) antibodies. Briefly, Petals at onset of flower opening were fixed in 1% (v/v) formaldehyde. Petal tissues were homogenized and nuclei were isolated and lyzed. Cross-linked chromatin was sonicated using a water bath Bioruptor UCD-200 (Diagenode, Liège, Belgium) (30 s on/30 s off pulses, at high intensity for 60 min). Protein/DNA complexes were immunoprecipitated with antibodies, overnight at 4°C with gentle shaking, and incubated for 1h at 4°C with 50 μL of Dynabeads Protein A (Invitrogen, Ref. 100-02D). the beads were wash 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 250μL Elution Buffer (1% SDS, 0.1 M NaHCO3). Chromatin was reverse-crosslinked by adding 20μL 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 20μg of glycogen and resuspended in 20μL 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 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.
identifiers.namespace: SUB3121829
identifiers.id: RcHt_H3K27me3_mark_Petal