GSM3038935: plum_4wk_SUL_3; Prunus domestica; OTHER
Frozen tissue was homogenized in Polysome Extraction Buffer (PEB; 200 mM Tris-HCl, pH 9.0, 200 mM KCl, 25 mM ethylene glycol tetraacetic acid (EGTA) pH 8.0, 35 mM MgCl2, 1% (v/v) octylphenyl-polyethylene glycol (Igepal CA-630), 1% (v/v) polyoxyethylene 10 tridecyl ether, 1% (v/v) sodium deoxycholate, 5 mM dithiothreitol (DTT), 1 mM PMSF, 50 ug/mL cycloheximide, 50 ug/mL chloramphenicol, 0.5 mg/mL heparin) using 10 mL PEB per 5 g of tissue. Homogenates were clarified by centrifugation at 16,000 g for 15 min and filtrated with cheesecloth. The supernatant was loaded onto an 8 mL 1.6 M sucrose cushion. Samples were ultracentrifuged at 170,000 g for 18 h at 4 C to pellet polysomes. 1 mL of PEB was used to resuspend the pellet. 50 μL of anti-FLAG magnetic beads (Sigma Chemical Company, St. Louis, MO USA) were added to the resuspended pellet and incubated at 4C overnight with gentle rocking. The beads were recovered using a magnet and washed four times for 5 min with 5 mL of wash buffer (200 mM Tris-HCl, pH 9.0, 200 mM KCl, 25 mM EGTA, 35 mM MgCl2, 5 mM DTT, 50 μg/mL cycloheximide, 50 μg/mL chloramphenicol). The complexes were eluted by treatment of the magnetic beads with 100 uL of Elution Buffer (100 uL wash buffer, 10 uL of 5mg/mL FLAG3 peptide - Sigma Chemical Company, St. Louis, MO USA, 0.5 uL of 2 U/mL RNase OUT - Thermo Fisher Scientific Cleveland, Ohio, USA). RLT buffer plus 2-Mercaptoethanol from the Qiagen RNeasy kit (Qiagen, Valencia, CA USA) was added to the eluted complexes. A 0.5 X volume of 100% ethanol was then added and the sample transferred to Qiagen RNeasy columns. Washes and RNA elutions were performed according to the manufacturer's instructions. Isolated RNA was measured on a NanoDrop 1000 and the quality of the RNA confirmed using a 2100 Bioanalyzer Eukaryotic Total RNA Nanochip (Agilent Technologies Palo Alto, CA USA). Library preparation and paired-end sequencing on the Illumina HiSeq system was done by Genewiz (South Plainfield, NJ, USA).
GEO Accession
GSM3038935