GSM8121175: Polysome_WT_Rep5; Mus musculus; RNA-Seq
GNPs were isolated, and cell pellet snap frozen in liquid N2 (LN2). The 1.5 ml Eppendorf tubes containing the GNP pellet was placed in the mortar super cooled under LN2. 150 ml of 1X polysome lysis buffer- pH 7.5 (10 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Nonidet-P40, 10 mM DTT, 100 µg/ml cycloheximide, 1 U/ml SUPERaseIn RNase Inhibitor, 1X protease inhibitor cocktail (EDTA-free) in RNAse-free deionized water). The frozen mix of cell pellet and lysis buffer was pulverized using a pre-chilled pestle. Eppendorf tubes were placed on ice to thaw and mixed with pipetting using a 200 ml tip. Samples were centrifuged at 2000×g for 5 min at 4°C to pellet nuclei and cell debris. Supernatant was transferred to a fresh tube and centrifuged at 12,000×g for 5 min at 4°C. 150 ml of the resulting supernatant was loaded onto the top of a 10-50% sucrose gradient in 4 ml Polypropylene ultracentrifuge tubes (Cat# 344062, Beckman Coulter, Inc) . The tubes were centrifuged in a SW60Ti rotor at 246,606×g (38,000 RPM) for 125 min with low acceleration and brakes off. All tubing was washed with RNase-free H2O at 3 ml/min using a clean ultracentrifuge tube. The BioComp™ Piston Gradient Fractionator was used to fractionate and record absorbance of the samples, setting the baseline absorbance by 60% sucrose. The first material to come out was free RNA and protein, followed by the 40S, 60S, 80S monosome, and increasing polysome peaks. Fractions were collected into microcentrifuge tubes as they exit the reader for use in downstream applications. Once the last of the sample was collected, several additional milliliters of 60% sucrose were flown through the tubing to clean it out and set the baseline again for next sample. RNA was extracted separately from monosomic, disomic and polysomic fractions using TRIzol™ Reagent (Life Technologies) and processed for RNA-sequencing. RNA libraries for RNA-seq were prepared following manufacturer's protocols