title: GSM6170912: PMC_H3K27me3_rep1; Oryza sativa; ChIP-Seq
library_construction_protocol: 8-10K cross-linked PMC pellet was suspended in pre-cooled cell lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.6% NP-40) for lysis on ice for 10min. After centrifuge at 12,000 rpm at 4℃ for 5min, the cell pellet was resuspended in the cell lysis buffer for additional 5 min incubation on ice. After washing twice using 1xMNase digestion buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2), the lysates were trimmed using MNase (Cat# M0247S, New England Biolabs) for 10 min at 37 °C. After adding 2x stopping buffer (100 mM Tris-HCl, pH 8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), MNase trimmed chromatin was sonicated for 10 cycles (20s on, 40s off). After centrifuge twice at 12,000 rpm at 4℃, 5min each, the clean supernatant was incubated with anti-H3K27me3 (Cat# 9733, Cell Signaling Technology) or anti-H3K4me3 (Cat#ab8580, abcam) with constant rotation at 4℃ overnight. The remaining procedures for recovery of ChIPed DNA were conducted following the published procedures (Saleh et al. 2008). Each antibody was biologically replicated. Biologically replicated ChIP-seq and input libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB, E7645S). All prepared libraries were sequenced on the Illumina NovaSeq platform.