GSM7660922: 16 DAP END, Input, Replicate 3; Hordeum vulgare; ChIP-Seq
Approximately 2 g of 16 DAP endosperm tissue were isolated and cross-linked under vacuum in 1% formaldehyde for 15 min at room temperature. The cross-linking was quenched by adding glycine to a final concentration of 0.1 M, and the vacuum was applied for 5 min. Endosperm tissue was rinsed with water and frozen in liquid nitrogen. Isolated nuclei were resuspended in Nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5% SDS, cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche)) and incubated at 4°C under gentle agitation for 20 min. The chromatin solution was sonicated using a Biorupter Plus (Diagenode) with 10 cycles of 30 sec pulse/90 sec cooling at high power at 4°C. The resulting sheared chromatin was pooled and diluted 1:4 with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.25 mM EDTA, 1.25% Triton X-100, 1 cOmplete™, EDTA-free Protease Inhibitor Cocktail). 600 µl aliquots of diluted chromatin were incubated with 7 µl of the Anti-trimethyl-Histone H3 (Lys27) Antibody (Millipore, cat. no. 07-449) in a rotator at 4°C overnight. Samples without antibody were used as negative controls. The next day, 40 µl of the Dynabeads Protein A (Invitrogen) were added to each tube, and the samples were further incubated for 2 hours. Afterward, beads were washed with a sequence of buffers, and immune complexes were eluted as described. Control chromatin aliquots ('input DNA') were taken prior to immunoprecipitation. Reverse crosslinking was performed for all samples, and  DNA was extracted and purified using the ChIP DNA Clean & Concentrator™ kit (Zymo Research). Sequencing libraries were prepared and 150-bp pair-end reads were sequenced using Illumina NovaSeq 6000 platform (Illumina) by Novogene Ltd.