GSM9144061: ChIPseq_SetB_antiH3K4me3_rdr6_rep1; Arabidopsis thaliana; ChIP-Seq
All the material for ChIP used in this paper were from pooled 10-12 day old seedlings grown on Murashinge and Skoog agar plates (1/2X MS, 1.5% agar, pH5.7). 2-4g of seedling tissue was used for each sample. Samples were ground in liquid nitrogen and crosslinked for 10 or 12 minutes at room temperature in Nuclei Isolation buffer (50mM Hepes pH8, 1M sucrose, 5mM KCl, 5mM MgCl2, 0.6% triton X-100, 0.4 mM PMSF, 5 mM benzamidine hydrochloride, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)) containing 1% formaldehyde. The crosslinking reactions were stopped with 125 mM glycine by incubation at room temperature for 10 minutes. 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 (Villar and Kohler, 2018)). 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 antibody. 7 µl of anti-HA (3F10, Merck) and 5 µl of anti-H3K4me3 (ab8580, abcam) were added to up to 2 ml of sheared chromatin per sample for pulldown. . 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, for 5 minutes each at 4°C (buffer compositions are described in the published protocol (Villar and Kohler, 2018)) and eluted twice with 250 µl of elution buffer (1% SDS and 0.1 M NaHCO3) in a thermomixer at 65°C and 1000 rpm, 20 minutes for each elution. 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. For the RNA-seq, plant material was extracted using the Direct-zol RNA miniprep kit (Zymo R2052) according to manufacturers instructions, including the in column DNase I treatment. ChIP-seq libraries were generated using NuGen Ovation Ultra Low System V2 kits according to manufacturer's instructions. Quantseq libraries were prepared using QuantSeq 3' mRNA-Seq Library Prep Kit FWD (Lexogen) according to manufacturer's instructions.