GSM5026053: ChIP_lib2_Col0_FLAG; Arabidopsis thaliana; ChIP-Seq
The experimental procedure was performed as described in PMID 20734276 with some variations. 2 or 2.5 g of inflorescences were used for each ChIP sample. Samples were ground in liquid nitrogen and crosslinked for 10 or 12 minutes at room temperature in Nuclei Isolation buffer containing 1% formaldehyde.  Crosslinked nuclei were filtered through one layer of miracloth, washed with Extraction Buffer 2, centrifuged through a layer of Extraction Buffer 3 and lysed with Nuclei lysis buffer (buffer compositions are described in the published protocol). Chromatin was sheared using a Bioruptor Plus (Diagenode) (20 cycles of 30 seconds on and 30 seconds off) and immunoprecipitated overnight at 4°C with 3.5 µl of Anti-FLAG M2 (Sigma, F1804). 25 µl each of Protein A and Protein G magnetic Dynabeads (Invitrogen) were added to each sample and incubated for 2 more hours at 4°C. The immunoprecipitated chromatin was washed with Low Salt (2X), High Salt, LiCl and TE buffers and eluted with elution buffer (1% SDS and 0.1 M NaHCO3). Reverse crosslinking was performed overnight at 65°C in 0.2 M NaCl, and proteins were degraded by proteinase K treatment at 45°C for 5h. The DNA fragments were purified using phenol:chloroform and ethanol precipitated overnight at -20°C. Libraries were prepared using the Ovation Ultra Low System V2 1-16 kit (NuGEN) following the manufacturer's instructions, with 15 cycles of PCR.