title: GSM3820394: pSUC2_Control_6wk_4; Prunus domestica; RNA-Seq
library_construction_protocol: 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 dithiothereitol (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. Samples were centrifuged at 16,000 g for 15 min at 4°C. The supernatant was loaded onto an 8 mL 1.6 M sucrose cushion. The polysome fraction was pelleted by centrifuging the samples at 170,000 g for 18 h at 4 °C. The pellet was resuspended in 1 mL of PEB. 50 μl of Anti-FLAG magnetic beads (Sigma Chemical Company, St. Louis, MO, USA) was added to the resuspended pellet and incubated at 4°C overnight with gentle rocking. The beads were recovered using their magnetic property and were washed four times in 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). Polysome-RNA complexes were eluted by treatment of the magnetic beads with 100 μl of Elution Buffer (100 μl wash buffer, 10 μl of 5 mg/mL FLAG3 peptide—Sigma Chemical Company, St. Louis, MO USA, 0.5 μl of 2 U/ml RNAse OUT—Thermo Fisher Scientific Cleveland, OH, USA). RLT buffer plus β-mercaptoethanol from the Qiagen RNeasy kit (Qiagen, Valencia, CA, USA) was added to the eluted complexes. A 0.5 × 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 2000 and with the Qubit RNA Assay Kit (Thermo Fisher Scientific, Cleveland, OH, USA). Quality of the RNA was analyzed using an RNA ScreenTape assay (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).
attributes.tag: GEO Accession
attributes.value: GSM3820394