GSM5026055: ChIP_lib3_SLN_FLAG_mbd56_bkg; 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  minutes at room temperature in Nuclei Isolation buffer containing 1.5 mM ethylene glycol bis(succinimidyl succinate) (EGS) and subsequently for another 10 minutes with 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.