GSM5605681: ChIP-Seq EPL_2_T; Arabidopsis thaliana; ChIP-Seq
RNA for each biological repeat was extracted from 110 mg of rosette leaves number 5-6 (from at least six plants) with TRI Reagent (Merck) and rounds of phenol-chloroform and chloroform extractions followed by isopropanol precipitation. RNA was treated with RQ1 DNase I (Promega), then extracted with phenol-chloroform and precipitated with ethanol. Chromatin was isolated as previously described (Sura et al. Plant Cell 2017), enriched with 2% chromatin isolated from mouse embryonic fibroblasts as spike-in control, and incubated with antibody (α-H3, Abcam ab1791; α-H4K5ac, Millipore 07327; α-H3K9ac Millipore 07352; α-HTA9, Agrisera AS10718; α-H2A.Zac, Diagenode C15410173 pAb-173-050) overnight at 4°C. Dynabeads Protein A (Thermo Fisher Scientific) were added to the samples and incubated for another 2 hours at 4°C. The slurry was washed twice with low salt buffer (with 150 mM NaCl), twice with high salt buffer (with 500 mM NaCl) and once with LiCl wash buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.25 M LiCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM PMSF and protease inhibitor cocktail). Chromatin was eluted through incubation in 1% SDS and 0.8 M NaHCO3 at 65°C and the cross-links were reversed by overnight incubation at 65°C. Following Proteinase K treatment and phenol/chloroform extraction, DNA was precipitated and DNA concentration was measured with Qubit dsDNA HS Kit (Thermo Fisher Scientific). Pure RNA solution was sent to Macrogen Europe (South Korea), where libraries were performed using TruSeq Stranded Total RNA sample preparation with Ribo-Zero Plant. Libraries were sequenced on the Illumina HiSeq 4000, 2x100bp run. 10 ng (H3) or 2 ng (other antibodies) of ChIP-ed DNA was used to prepare DNA libraries with MicroPlex Library Preparation Kit v2 (Diagenode). The library preparation was carried out accoring to the manufacturer (Diagenode) protocol.