GSM5777413: H3K36me3_sdg26;aprf1_rep1; Arabidopsis thaliana; ChIP-Seq
A 2-g quantity of 10-day-old seedlings was harvested and ground into a fine powder in liquid nitrogen. The powder was suspended in 15 ml of Nuclear Extraction Buffer (NEB) (20 mM Tris-HCl [pH 7.5], 20 mM KCl, 2 mM EDTA [pH 8.0], 2.5 mM MgCl2, 25% glycerol, 250 mM sucrose, 5 mM DTT, 1% protease inhibitor cocktail, and 1 mM PMSF) and was then subjected to cross-linking with 1% formaldehyde (Sigma, F8775). The nuclei were washed in Nuclear Resuspension Buffer (NRBT) (20 mM Tris-HCl [pH 7.5], 2.5 mM MgCl2, 25% glycerol, and 0.2% Triton X-100) and then sonicated in Sonication Buffer (20 mM Tris-HCl [pH 8.0], 2 mM EDTA [pH 8.0], 0.2% NP-40, 1 mM PMSF, and 1% PPI). The sheared chromatin was diluted with Dilution Buffer (2 mM EDTA, 20 mM Tris-HCl [pH 8.0], 200 mM NaCl, 1 mM PSMF, and 1% protease inhibitor cocktail) and incubated with GBP (GFP-binding protein) coupled with  agarose beads (Cytvita, 17-096-01) or incubated with H3Ac antibody (Millipore, 06-599), H3K4me3 antibody (Abcam, ab8580), H3K27me3 antibody (Millipore, 07-449), and H3K36me3 antibody (Abcam, ab9050) coupled with Dynabeads Protein A (Invitrogen, 100–01D) at 4℃ overnight. The beads were washed 5 times with Wash Buffer (150 mM NaCl, 20 mM Tris-HCl [pH 8.0], 2 mM EDTA [pH 8.0], 0.1% Triton X-100, and 1 mM PMSF) and then with TE (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA [pH 8.0]). The bead-bound DNA-protein complexes were eluted from the beads and reverse cross-linked with NaCl and Proteinase K (Sigma, P4850). The chromatin DNA was extracted using Phenol/Chloroform/Isoamyl alcohol and was then subjected to qPCR or library construction. Libraries were built for ChIP-seq (Illumina NovaSeq 6000; 150-bp paired-end sequencing)