GSM5461913: Monoecious WT H3K27me3 ChIP-seq  rep1; Cucumis melo; ChIP-Seq
1g of fresh leaves was crosslinked in 1% (v/v) formaldehyde for 15min. Crosslinking was then stopped by adding glycine in a final solution of 0.25M solution for 5min. Samples were homogenized, and nuclei was isolated and lysed. Cross‐linked chromatin was sonicated using a Covaris S220 (Peak Power: 175, cycles/burst: 200. Duty Factory: 20) for 9 min. Protein-DNA complexes were immunoprecipitated with rabbit anti-H3K27me3 (Millipore, ref. 07-449)  antibodies overnight at 4 °C with gentle shaking and later incubated for 2h at 4 °C. Posteriorly, for H3K27me3 IP, chromatin-antibody complexes were incubated for 50 μL of Dyna- beads Protein A (Invitrogen, Ref. 100-02D). Beads were washed for 2 × 5 min in ChIP Wash Buﬀer 1 (0.1% SDS, 1% Triton X-100, 20mM 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‐cross‐linked by adding 20 μl of NaCl 5M and incubated overnight at 65°C. Reverse cross‐linked DNA was then treated with RNase and proteinase K digestion, and extracted with phenol−chloroform. DNA was ethanol precipitated in the presence of 20 μg of glycogen and incubated overnight at -20°C. DNA pellet was then resuspended in 20 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 NEB-Next Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's recommendations. NEBNext Ultra II FS DNA Library Prep Kit for Illumina (E5745S)