GSM6323625: 100 varieties, mix; Triticum aestivum; DNase-Hypersensitivity
1cm-long-leaves of the plants were harvested, mixed together and fixed with 1% formaldehyde in HEPES buffer (50 mM HEPES, 1 mM EDTA, pH 8.0, 0.1 M NaCl, and 1 mM PMSF). The fixed leaves were ground to a fine powder in liquid nitrogen. Wheat nuclei were extracted with H1B buffer (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, 5 mM spermidine, 0.15 mM spermine, 40% glycerol, and 0.1% mercaptoethanol). The extracted nuclei were purified with H1B buffer supplemented with 0.5% Triton X-100. The purified nuclei were washed once with RSB buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, and 3 mM MgCl2). The pelleted nuclei were resuspended with 2 ml RSB buffer and then divided equally into five 1.5-ml Eppendorf tubes. The aliquoted nuclei were digested with DNase I (0, 0.01, 0.03, 0.05, and 0.08 units). The resulting digested nuclei were extracted using 1 volume of phenol, phenol:chloroform, and chloroform, after which the DNA from each digestion was resuspended in 2 volumes of cold ethanol and then pelleted. A DNase-sequencing library was prepared from 0.03 U DNase I-digested nuclei. Approximately 2 µg DNA was separated by 1.5% agarose gel electrophoresis, and DNA fragments (50-300 bp) were cut and purified to prepare the DNase-sequencing library with the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB).