GSM6622816: RNA_frs7 frs12_rep1; Arabidopsis thaliana; RNA-Seq
Total RNA from 5-day seedling was extracted with a RNeasy Plant Mini kit (Qiagen). For RNA-seq analysis, the total RNA was treated with a Poly(A) mRNA Magnetic Isolation Module Kit (NEB #E7490). DNA of 6th, 7th and 8th leaves was purified using a DNeasy® Plant Mini Kit (Qiagen) and eluted in 150 µL of elution buffer. Experiments were performed for two biological replicates.  The nuclear matrix was isolated as previously described in (53). Briefly, Arabidopsis 5-day-old whole plants grown under LD condition (16h at 23 °C under 22 μmol·m−2·s−1 continuous white light, 8h at 23 °C dark with 70% humidity) were collected and chopped in 1 ml LB01 buffer (2 mM Na2EDTA, 20 mM NaCl, 2 mM EDTA, 80 mM KCl, 0.5 mM spermine, 15 mM β-mercaptoethanol, 0.1% Triton X-100, pH 7.5). Another 9 ml LB01 buffer was used to wash the nuclei into 1-layer miracloth filter followed by a one-time filter through 30 μm CellTrics (Sysmex, Germany, 04-0042-2316). The nuclei mixture was centrifuged at 1500g for 10 min at 4 °C. The nuclei pellet was further wash with DNase I buffer (20 mM Tris pH 7.4, 20 mM KCl, 70 mM NaCl, 10 mM MgCl2, 0.125 mM spermidine 1 mM PMSF, 0.5% Triton-X 100) until white. After centrifuge, the nuclei were resuspended in 200 μl DNase I buffer, and an aliquot of nuclei was stored for isolation and estimation of total genomic DNA as control. DNase I (Thermo Scientific, EN0525) was added and incubated at 4°C for 1 hr. Nuclei were collected by centrifugation at 3000g for 10 min at 4 °C. Digestion was followed by extraction with 0.4 M NaCl for 5 min twice on ice in Extraction Buffer (10 mM Hepes pH 7.5, 4 mM EDTA, 0.25 mM spermidine, 0.1 mM PMSF, 0.5% (v/v) Triton X-100). Another two times extraction with 2 M NaCl for 5 min on ice in Extraction Buffer. The final nuclear matrix pellet was washed twice with Wash Buffer (5 mM Tris pH 7.4, 20 mM KCl, 1 mM EDTA, 0.25 mM spermidine, 0.1 mM PMSF). RNA-seq Libraries were prepared from the resulting mRNA with a NEBNext Ultra Kit (NEB #E7530). The libraries were constructed according to a standard protocol (Illumina). The RNA-seq libraries were sequenced at Novogene via a Novaseq instrument in 150-bp paired-end mode.  MAR-seq libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645) according to the manufacturer's instructions. The libraries were sequenced at Novogene (UK) via a Novaseq instrument in 150-bp paired-end mode. Two biological replicates were performed.