GSM8987639: imbibed input rep2; Arabidopsis thaliana; ChIP-Seq
250 mg of dry and 6-hour-imbibed seeds were ground in liquid nitrogen. The resulting powder was combined with 20 ml of crosslinking buffer (0.1 M sucrose; 0.1 M NaCl; 20 mM HEPES pH 7.0; 1% formaldehyde) in 50 ml tubes and gently mixed in the cold room for 10 minutes. Subsequently, 1.25 ml of 2 M glycine was added to each sample and mixed gently in the cold room for 5 minutes. The samples were then centrifuged at 1800 g for 15 minutes at 4°C, after which the supernatant was removed, and 20 ml of Honda buffer (0.44 M sucrose; 1.25% Ficoll; 2.5% Dextran 40; 25 mM HEPES pH 7.0; 10 mM MgCl2; 0.5% Triton X-100; 0.5 mM PMSF; 10 mM DTT; 1x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)) was added to the pellet, which was then resuspended by mixing with a spatula. The samples were mixed gently in the cold room for 10 minutes and filtered through two Miracloth layers. They were then centrifuged at 1800 g for 15 minutes at 4°C; the supernatant was removed, and the pellet was resuspended in 500 µl of fresh NLB buffer (50 mM Tris-HCl pH 8.0; 10 mM EDTA; 1% SDS; 1x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)), after which the samples were incubated on ice for 30 minutes. Next, 500 µl of ChIP dilution buffer I (16.7 mM Tris-HCl pH 8.0; 167 mM NaCl; 1.2 mM EDTA) was added to the samples. The samples were sonicated using a Bioruptor (Diagenode) with internal cooling, employing 30 seconds ON/30 seconds OFF (High) settings for 20 cycles with 5-minute breaks for cooling on ice after every 5 cycles. The samples were centrifuged for 5 minutes at 20,000 g and 4°C, with the supernatants being carefully transferred to new tubes to avoid contamination from the upper layer. 60 μl of each sample were retained as input controls. Immunoprecipitation was conducted in high-recovery tubes using 400 µl of chromatin, to which 1600 µl of ChIP dilution buffer II (16.7 mM Tris-HCl pH 8.0; 167 mM NaCl; 1.2 mM EDTA; 1.1% Triton X-100; 0.6 mM PMSF; 1.2x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)) was added. The IP was performed with 2.5 µg of anti-RPB1 antibody (Agrisera AS11 1804), and the samples were incubated in the cold room with gentle rotation. The next day, 15 µl of ChIP dilution buffer II-washed Dynabeads Protein G (ThermoFisher) were added to the samples and incubated in the cold room with gentle rotation for 3 hours. The supernatant was discarded, and the beads were washed twice with low salt buffer (150 mM NaCl; 0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl pH 8.0), once with high salt buffer (500 mM NaCl; 0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl pH 8.0), and twice with TE buffer. For ChIP-seq, antibody complexes were eluted by adding 100 µl of elution buffer (1% SDS; 0.1 M NaHCO3) to the beads and incubating at 65°C for 15 minutes. The supernatant was collected, and the elution was repeated with a fresh 100 µl of elution buffer. The supernatants were combined, mixed with 8 µl of 5M NaCl, and incubated overnight at 65°C. Input samples were processed similarly. To the input samples, 100 µl of TE buffer, 7.1 µl of 5 M NaCl, and 17.4 µl of 10% SDS were added, followed by an overnight incubation at 65°C. The following day, 5 µl of 0.5 M EDTA, 8 µl of 1 M Tris-HCl pH 6.5, and 2 µl of 20 mg/ml proteinase K were added to all samples, which were then incubated for 3 h at 55°C. DNA was recovered using ChIP DNA Clean & Concentrator columns (Zymo). Libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina.