title: GSM6285417: SYD-GFP spt6ldeltatSH2 rep2; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: plants were crosslinked with 1% formaldehyde for 20 min under vacuum at room temperature and quenched with 0.125 M glycine under vacuum for an additional 5 min. Approximately 500 milligrams of tissues were used for each ChIP assay. The chromatin was sonicated using a Covaris M220 sonicator (peak power: 75; duty factor: 20; cycles/burst: 200) for 210 s at 6°C. The lysates were centrifuged at 18,000 g for 10 min at 4°C to remove debris. 10 µL of supernatant was used as the input sample, whereas the remaining supernatant was kept for the downstream procedure. 10 µL of Dynabeads Protein A (Cat. 10002D, ThermoFisher SCIENTIFIC) were added to each sample to prewash for 1 h at 4°C with gentle rotating. ChIP was carried out using antibodies as follows: anti-GFP (Cat. ab290, Abcam) and anti-Pol II CTD repeat YSPTSPS (Cat. ab817, Abcam). 40 µL of Dynabeads Protein A were mixed with 4 µL of antibodies and incubated for approximately 6 h on a rotator at 4°C to form antibody-bead complexes. Finally, ChIP-DNA were recovered by Phenol/chloroform extraction. Approximately 400 milligrams of seedlings were used per sample. Additionally, equal amounts of spike-in nuclei extracted from Saccharomyces cerevisiae were added to each sample before MNase treatment, which worked as the internal control among samples. For MNase treatment, the prepared nuclei were resuspended in prewarmed MNase digestion buffer, followed by the addition of 8 units of MNase (Cat. 2910A, TaKaRa) and incubation (15 min at 37°C with periodic agitation). Finally, the mono-nucleosomal DNA was isolated from 2% agarose gels and quantified by the Qubit dsDNA HS assay kit (Cat. Q32851, ThermoFisher SCIENTIFIC). ChIP libraries were prepared by using the NEBNext® Ultra™ DNA Library Prep Kit (E7370S) following the manufacturer's instructions, and subsequently used for Illumina