GSM2574857: Nb_p35S_Mock1; Nicotiana benthamiana; RNA-Seq
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.  Homogenates were clarified by centrifugation at 16,000 g for 15 min and filtered 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. Anti-FLAG magnetic beads, 50 μL (Sigma Chemical Company, St. Louis, MO USA) were added to the resupsended pellet and incubated at 4oC 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). cDNA libraries were prepared by the by the UM-IBBR Sequencing Core at University of Maryland, College Park (http://ibbr.umd.edu/facilities/sequencing) using the Illuminia TrueSeq RNA library kit according to the manufacturer's instructions.