GSM5218418: WT-RNAseq-rep-3; Arabidopsis thaliana; RNA-Seq
ChIP seq: Samples were crosslinked in vitro. Nuclei were prepared with Nuclei Isolation Buffer (50 mM HEPES, 1 M sucrose, 5 mM KCl, 5 mM MgCl2, 0.6% Triton X-100, 0.4 mM PMSF, 5 mM benzamidine, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)). Chromatin was sheared with Bioruptor Plus (Diagenode) for 20 minutes with 30 s on and 30 s off and incubated with anti-FLAG M2 (Sigma) antibody overnight at 4◦C. Chromatin bound proteins were immunoprecipitated with Protein A and Protein G magnetic Dynabeads (Invitrogen) for 2 hours at 4◦C. DNA fragments were precipitated with EtOH overnight at -20◦C. Libraries were prepared with Ovation Ultra Low System V2 kits following the manufacturer's instructions. BS seq: DNA from floral tissues were extracted with cetyl trimethylammonium bromide (CTAB)-based method. RNA was removed with PureLink RNase A (Invitrogen). 300 ng of DNA was sheared to 200bp with a Covaris S2 (Covaris). RNA seq: Floral tissues from 5-6 week old seedlings were used for RNA extraction with Zymo Direct-zol RNA MiniPrep kit (Zymo Research) following manufacturer's instructions. ChIP seq: Libraries were prepared with Ovation Ultra Low System V2 kits following the manufacturer's instructions. BS seeq: 300 ng of DNA was sheared to 200bp with a Covaris S2 (Covaris). Libraries were prepared with the Epitect Bisulfite Conversion kit (QIAGEN) and the Ovation Ultralow Methyl-seq kit (NuGEN) following the manufacturer's instructions. RNA seq: 1 ug of total RNA was used for library preparation with TruSeq Stranded mRNA kit (Illumina). Libraries were sequenced on HiSeq 2500 or NovaSeq 6000 (Illumina). RNA seq: Leaf tissues from three-week-old seedlings were used for RNA extraction with Zymo Direct-zol RNA MiniPrep kit (Zymo Research) following manufacturer's instructions. 1 ug of total RNA was used for library preparation with TruSeq Stranded mRNA kit (Illumina). Libraries were sequenced on HiSeq 2500 or NovaSeq 6000 (Illumina).