Total RNA Extraction and poly(A)+ RNA Preparation

Cells were broken by standard procedures with the following modifications. Yeast cell pellets were re-suspended in breaking buffer (50 mM Tris-Cl, pH 8, 100 mM NaCl, 10 mM EDTA, 5% SDS) and vortexed for 5 min on a multitube vortexer at maximum setting in the presence of 30% glass beads (425–600 μm mesh; Sigma) and 30% phenol/chlorophorm/isoamylalcohol (25:25:1, v/v/v). After centrifugation the aqueous phase was reextracted and ethanol precipitated. The nucleic acid pellets were resuspended in guanidine isothiocyanate lysis buffer. Total RNA was extracted by cesium chloride ultracentrifugation. The poly(A)⁺ RNA was prepared using the FastTrack 2.0 Kit (Invitrogen, Leek, Netherlands). The absence of mRNA degradation was assessed by visualization of residual ribosomal 28S and 18S mRNAs on a denaturing agarose gel.