GSM5655312: pds5_H3K9me2_rep1; 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) . For ChIP-seq library preparation, closed buds were collected. Chip assay from cross-link to chromatin shearing was previously described by Lambing, et al (Lambing et al. 2020) with minor modifications. Briefly, the materials were firstly collected in liquid nitrogen and grind to fine powder. Nuclei isolation buffer (1M sucrose, 5mM KCl, 5mM MgCl2, 5 mM EDTA, 60 mM Hepes pH 8.0, 0.6% Triton X-100, 0.4 mM PMSF, 1 μM pepstatin A, 1X Protease Inhibitor Cocktail) and 1% final concentration formaldehyde solution were added to each sample. Incubating the sample on a Thermo roller mixer (cat number) at 12 rpm for 25 min for closed buds or 10 min for seedlings at room temperature. Cross-link was stopped by adding glycine to a final concentration of 130 mM for another 25 min for closed buds. After the isolation of nuclei, the chromatin was sonicated with Nuclei lysis buffer (50mMTris–HCl, pH 8.0, 10 mM EDTA, 1% SDS, 0.1 mM PMSF, 1 μM pepstatin A, Protease Inhibitor Cocktail) to obtain the desired DNA fragment (size enriched at 300 bp) by a Bioruptor® Plus sonication device. The following procedures were described by Moreno-Romero et al (Moreno-Romero et al. 2016). After the de-crosslinking, DNA was purified according to the manual of IPure kit v2 Kit (C03010015, Diagenode). Chip-seq libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit (NEB #E7645). The antibodies we used: anti-H3 (H9289, Sigma), anti-H3K9me2 (C15410060, Diagenode) and anti-GFP (A11122, Invitrogen). The library was sequenced on an Illumina Novaseq instrument with 2 × 150 bp reads.. Libraries were prepared for sequencing using standard Illumina protocols