Determination of ncRNAs repressed by Isw2

It has previously been shown that Isw2 represses cryptic antisense transcripts from the 3’-end of three genes. However, whether Isw2 generally functions to repress cryptic RNA transcription is currently unknown. We thus sought to determine the loci at which Isw2 is required to repress cryptic non-coding RNA (ncRNA) expression and to map their transcription start sites relative to nucleosome positions on a global scale. We hybridized total RNA from Isw2 deletion strains to high-resolution, strand-specific microarrays tiling chromosomes III, VI, and XII, covering ~14% of the genome. Because the exosome complex efficiently degrades cryptic transcripts and are not expected to alter the frequency of ncRNA transcription, deletion of components in the exosome pathway, either TRF4 or RRP6, in combination with ISW2 (Δisw2 trf4 and Δisw2 rrp6, respectively) is required to stabilize some cryptic transcripts. Furthermore, because it is unclear how cryptic RNA transcripts are processed in vivo, especially in the absence of Trf4 or Rrp6, we avoided any selection or amplification of RNA. At least three replicates (including at least one biological and one technical replicate) each of WT, Δisw2, Δtrf4, Δrrp6, Δisw2 trf4 and Δisw2 rrp6 were analyzed.