GSM5606739: 2xCol_H3_rep1; Arabidopsis thaliana; ChIP-Seq
For RNA-seq library preparation, total RNA from two-week-old seedlings was extracted with a RNeasy Plant Mini kit (Qiagen). Afterwards, the total RNA from seedlings was treated with a Poly(A) mRNA Magnetic Isolation Module Kit (NEB #E7490). Libraries were prepared from the resulting mRNA with a NEBNext Ultra Kit (NEB #E7530). For ChIP-seq library preparation, two weeks-old seedlings grown were collected. The ChIP experiments essentially followed a published protocol (Moreno-Romero et al., 2017) with minor changes. In brief, the chromatin was sheared to an average size of 350 bp with a Bioruptor® Plus sonication device. The sonicated sample was then divided into two and immunoprecipitated with 1μg of anti-H3 (Abcam ab1791) or 2.5μg of anti-H3K27me3 antibodies (07-449, Millipore). After overnight incubation at 4 °C, the antibodies were recovered with Dynabeads protein A (Invitrogen, cat. No. 10001D) followed by a series of washing steps as described (Moreno-Romero et al., 2016). An IPure kit v2 (Diagenode, Belgium) was used to purify the DNA. Afterward, the purifiedDNA was used for qPCR or library preparation. The steps of library preparation were performed with the NEBNext® Ultra™ II DNA Library Prep Kit (NEB). The library was sequenced on an Illumina Novaseq instrument with 2 × 150 bp reads.. RNA-seq Libraries were prepared for sequencing using standard Illumina protocols For ChIP-seq library preparation, the purified ChIP-ed DNA was utilized for the subsequent end repairing, A-tailing, adaptor ligation, and library amplification. The steps were performed with the NEBNext® Ultra™ II DNA Library Prep Kit (NEB).  The library was sequenced on an Illumina Novaseq instrument with 2 × 150 bp reads.