GSM8292138: ChIP_seq_WT; Arabidopsis thaliana; ChIP-Seq
The chromatin immunoprecipitation (ChIP) protocol was conducted according to previously described methods with minor adjustments (Diego-Martín et al. 2022). Briefly, 1 gram of inflorescences was ground in liquid nitrogen and cross-linked in Nuclei Isolation Buffer containing 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was quenched with glycine, followed by nuclei isolation and chromatin shearing using a Bioruptor Pico (Diagenode). Chromatin was then immunoprecipitated overnight at 4°C with Anti-FLAG M2 antibody (5 μl/ChIP, F1804, Sigma). The immunocomplexes were captured using a 1:1 mixture of magnetic Protein A and Protein G Dynabeads (Invitrogen) for 3 hours at 4°C, followed by sequential washing with low salt, high salt, LiCl, and TE buffers for 10 minutes each at 4°C. Elution of the immunoprecipitated complexes was performed for 2 × 20 minutes at 65°C with elution buffer. Reverse cross-linking was carried out overnight at 65°C, followed by proteinase K treatment at 45°C for 5 hours. DNA was purified using phenol:chloroform:isoamyl alcohol 25:24:1 (Fisher Scientific) and precipitated with GlycoBlue (Invitrogen) and NaAc/EtOH overnight at -20°C. The purified DNA was resuspended in 75 μl of elution buffer. Libraries for sequencing were prepared using the Ovation Ultra Low System V2 1–16 kit (NuGEN) following the manufacturer's instructions. Libraries were sequenced in a HiSeq 2500asSE50reads. Ovation Ultralow V2