GSM3819645: Myc BDR3 rep1; Arabidopsis thaliana; ChIP-Seq
The seedlings were crosslinked for 15 min in 1 % formaldehyde under vacuum (5 min vacuum / vacuum release / 10 min vacuum) followed by 6 min quenching with glycine (125mM final concentration, under vacuum), extensive rinsing with ice-cold water and drying. Crosslinked seedlings were ground into fine powder in liquid nitrogen, incubated for 15 min at 4°C in extraction buffer (25mM Tris-HCl, pH=8.0, 0.44M sucrose, 10mM MgCl2, 0.1 % Triton X-100, 2mM spermine, 10mM beta-mercaptoethanol, 1 mM PMSF, 1 % plant protease inhibitor cocktail) and filtered through 2 layers of Miracloth. Nuclei were prepared by 3 rounds of resuspension in extraction buffer (without spermine, twice with 0.25 % Triton X-100 and once with 0.1 % Triton X-100), incubation 12 min on ice and centrifugation at 1,000g for 12 min at 4°C (only 300g for the last centrifugation). The nuclei suspension was stored in 20 % glycerol at -70°C until used. After thawing on ice, nuclei were centrifuged for 10 min at 11,000g at 4°C and resuspended in nuclei lysis buffer (500 mM Tris-HCl, pH=8.0, 10mM EDTA, 1 % SDS, 1mM PMSF, 1 % plant protease inhibitor cocktail). Chromatin was sonicated for 12 min using a Covaris S220 (peak power=150, duty factor=20, cycles/burst=200). Soluble chromatin was recovered by centrifugation at 13,000g for 15 min at 4°C and diluted to (final concentrations): 20mM Tris-HCl, 150mM NaCl, 2mM EDTA, 0.1 % SDS, 1 % Triton X-100, 1mM PMSF, 1 % plant protease inhibitor cocktail. Libraries were prepared using NEBNext Ultra II DNA library prep kits (NEB E7645). Libraries were sequenced on a NextSeq 500 instrument at Indiana University Center for Genomics and Bioinformatics.