title: GSM6205323: H3K27me3; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: The chromatin immunoprecipitation protocol was done as previously described with minor changes (28). Briefly, 1 gram of inflorescences were ground in liquid nitrogen and fixed in Nuclei Isolation buffer containing 1% formaldehyde for 10 min at RT. In the case of CHR23-FLAG ChIPs, samples were first fixed with in Nuclei Isolation buffer containing 1.5mM EGS for 10 minutes at RT followed by addition of 1% formaldehyde and incubation for 10 additional minutes. Reactions were stopped with glycine followed by nuclei isolated and chromatin shearing using a Bioruptoptor pico (Diagenode). Chromatin was immunoprecipitated overnight at 4 °C with the following antibodies: Anti-FLAG M2 (5 ul/ChIP used, F1804, Sigma), Pol II Ser5 (5 ul/ChIP used, Ab5131), H3K4me3 (5 ul/ChIP used, 04-745, Millipore), H3K36me3 (10 ul/ChIP used, Ab9050, Abcam), H3K27me3 (10 ul/ChIP used, 07-449, Millipore), H3 (5 ul/ChIP used, Ab1791, Abcam), H2A (10 ul/ChIP used, Ab13923, Abcam), H2A.Z (3 ul/ChIP used), and PanH3Ac (5 ul/ChIP used, 39140, Active motif). Complexes were captured 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 × 20 min at 65 °C with elution buffer. Reverse 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 GlycoBlue (Invitrogen) and NaAc/EtOH overnight at −20 °C. DNA was resuspended in 75ul of elution buffer. Libraries for TPF2-FLAG and CHR23-FLAG experiments were prepared using the Ovation Ultra Low System V2 1-16 kit (NuGEN) following the manufacturer's instructions. The Nugen Ultralow V2 kit (TECAN) was used to make the libraries