GSM5918672: H3.1S28A, K27, rep2; Arabidopsis thaliana; ChIP-Seq
For RNA-seq library preparation, total RNA from closed buds was extracted with a RNeasy Plant Mini kit (Qiagen). Afterwards, the total RNA was treated with a Poly(A) mRNA Magnetic Isolation Module Kit (NEB #E7490). Libraries were prepared from the resulting mRNA with a VAHTS mRNA-seq V3 Library Prep Kit (Vazyme #NR611-01) .The ChIP experiments were performed as described by Moreno-Romero et al. (2017), with minor changes. In brief, after the isolation of nuclei, the chromatin was sonicated in Nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, 0.1 mM PMSF, 1 μM pepstatin A, Protease Inhibitor Cocktail (Roche, 11836145001)) using a Bioruptor® Plus sonication device (Diagenode) to obtain the desired DNA fragment size (enriched at 300 bp). The following procedures were described by Moreno-Romero [46]. After the de-crosslinking, DNA was purified according to the IPure kit v2 Kit manual (Diagenode, C03010015). ChIP-seq libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645). The following antibodies were used: anti-H3 (Sigma, H9289), anti-H3S28ph (Millipore, 07-145), and anti-H3K27me3 (Millipore, 07-449). Libraries were prepared for sequencing using standard Illumina protocols