High resolution tiling microarrays

Tandem affinity purifications of FTP-tagged strains were performed as described above with minor modifications. Reagents were prepared under RNase-free conditions and RNase inhibitor (Fermentas) was used during purification. IPed RNA–protein complexes were eluted from anti-Flag beads, followed by the phenol/chloroform/isoamyl alcohol extraction of RNAs. Total RNAs from the input were extracted using the TRI Reagent (Sigma Aldrich). The IPed RNAs and input RNAs were then treated using Turbo DNase (Life Technologies) to remove the potential DNA contamination. Reverse transcription was performed using the SuperScript Indirect cDNA labelling system (Life Technologies) with random hexamers. The cDNAs from IPed and input samples were labelled with Cy5 and Cy3 (GE Healthcare), respectively, and hybridized to high-resolution tiling microarrays (Agilent). RIP-Chip experiments were performed with at least two biological replicates.

WT and mutant strains were grown in YEA at 30 °C to OD₆₀₀ 0.5–0.8. Temperature-sensitive strains were grown in YEA at 23 °C to OD₆₀₀ 0.3–0.4 and then moved to 37 °C for 4 h (OD₆₀₀<0.8). Total RNAs from WT and mutants were isolated using the TRI Reagent (Sigma Aldrich), followed by rRNA depletion and reverse transcription using the SuperScript Indirect cDNA labelling system (Life Technologies) with anchored oligo(dT)₂₀ and random hexamers. cDNAs from WT and mutant strains were labelled with Cy3 and Cy5 (GE Healthcare), respectively, and hybridized to high-resolution tiling microarrays (Agilent) consisting of forward- and reverse-DNA-strand-specific probes. Expression arrays were performed with at least two biological replicates, except mmi1Δ and rmn1Δ.