title: GSM4785560: ChIP_H3_ZFSssI_line2; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: ChIPs were performed as described previously (GallegoBartolome et al.,2019). Briefly, 2 grams of inflorescences from untransformed Col-0 and T4 homozygous populations of two independent transgenic lines expressing ZF-SssI were ground in liquid nitrogen and fixed for 10 min in Nuclei Isolation buffer containing 1% formaldehyde. After stopping the reaction with glycine, nuclei were isolated, chromatin was sheared using a Bioruptor Plus (Diagenode) and immunoprecipitated overnight at 4°C with the following antibodies: Anti-FLAG M2 (F1804, Sigma), H3K4me1 (Ab8895, Abcam), H3K4me3 (04-745, Millipore), H3K36me3 (Ab9050, Abcam), H3K27me3 (07-449, Millipore), H3 (Ab1791, Abcam), H2A (Ab13923, Abcam), H2AZ (Ref Potok et al, Nat Comm, 2019), PanH3Ac (39140, Active motif). Chromatin-bound proteins were immunoprecipitated with a 1:1 mixture of magnetic Protein A and Protein G Dynabeads (Invitrogen) for 3 h at 4°C, washed with low salt, high salt, LiCl and TE buffers for 10 min each at 4°C and eluted for 2 x20 minutes at 65°C with elution buffer. Reversal crosslink was done overnight at 65°C, followed by proteinase K treatment at 45°C for 5 h. DNA was purified using Phenol:Chloroform:Isoamyl Alcohol 25:24:1 (Fisher Scientific) and precipitated with NaAc/EtOH and GlycoBlue (Invitrogen) overnight at −20°C. Libraries were prepared using the Ovation Ultra Low System V2 1-16 kit (NuGEN) following the manufacturer's instructions.