GSM2633782: Nuclear_RNA_Control_SAM; Oryza sativa Japonica Group; OTHER
Nuclei were purified from frozen and pulverized p35S:OsNTF2 tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including use of a 30 µm filter to exclude 30 to 70 µm cellular debris from the crude extract and extend centrifugation times. Tissue was resuspended in an ice cold mortar containing 10 mL of freshly prepared nuclei purification buffer (NPB: 20 mM MOPS, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine, 0.2 mM spermine, pH, 7.0) containing 200 uL Protease Inhibitor Cocktail (0.4X, Sigma, P9599) per 50 mL of buffer. The homogenized extracts were filtered through a 30 µM nylon mesh (Celltrics) to remove cell debris and centrifuged at 1000 x g for 15 min at 4° C to pellet nuclei. Nuclei were resuspended in 1 mL of NPB (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei in a cold room at 4° C for 30 min. The nuclei/beads suspension was diluted to 14 mL with NPB supplemented with 0.1% (v/v) Triton X-100 (NPB-T), in a 15 mL Falcon tube, mixed thoroughly and placed in a 15 ml magnet (adapted NEB 50 mL tube magnet) to capture bead-bound nuclei for 7 min at 4° C. The supernatant was carefully removed using a plastic Pasteur pipette, taking care to remove bubbles to avoid disturbing the beads. Beads were resuspended in 14 ml of cold NPB-T, placed on a rotating mixer for 30 sec, and then placed back in the 15 ml magnet to capture the beads-nuclei at 4° C for 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction. Nuclear RNA library, pre-rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was  increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre-rRNA degraded nuclear were prepared as described by Townsley et al. (2014)
GEO Accession
GSM2633782