GSM1570245: RNA-seq_control_2; Arabidopsis thaliana; RNA-Seq
To assess the effect of environmental stimuli on nucleosome occupancy, plants were treated with coronatine (COR) (Sigma, St. Louis, MO) or water (control) for 1 hr. The harvested samples were ground with liquid nitrogen and divided into 3 aliquots for nucleosome genomic DNA (gDNA), naked gDNA, and RNA isolation. For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes (pH 7.6), 5mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton-X 100, 0.4 mM phenylmethanesulfonyl fluoride, 1x ethylenediaminetetraacetic acid-free protease inhibitor (Roche, Mannheim, Germany)), digested with 0.4 U/μl micrococcal nuclease (MNase) (NEB, MA, USA) for 9 min, and purified with phenol/chloroform. To generate the naked gDNA sample, the gDNA was isolated without the nucleus isolation step as described previously (Dellaporta et al. 1983), purified with phenol/chloroform to strip off proteins, and digested with 0.25 U/μl MNase (NEB, MA, USA) for 1.75 or 3 min. Total RNA was purified with the E.Z.N.A. Plant RNA kit (Omega Bio-Tek, GA, USA) The purified nucleosome and naked gDNA was used to construct a library for 50-bp paired-end sequencing following the Illumina protocol. The purified total RNA was used to construct a library for 50-bp single-end sequencing following the Illumina protocol.