Cryptic Transcription in a set2∆ in Saccharomyces cerevisiae

In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution. Wild type (BY4741) and set2∆ (BY4741) strains were grown at 30°C in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD600 of 0.6-0.8. For each of the three replicates, Total RNA was extracted by acid-phenol method (Xiao et al. 2003). Double-stranded cDNA was prepared using an Invitrogen SuperScript™ (Cat No. 11917-010) primed with Oligo(dt) and random hexamers. For each replicate, the wt and set2∆ cDNA were independetly fluorescently labeled and comparatively hybridized to high-resolution 385K Saccharomyces cerevisiae CGH arrays (2005-08-16_SCER_WG_CGH) with Tm-normalized probes. In one of the replicates, assignment of the fluorescent label was reversed.