Cells were mixed in a defined ratio (1 S. cerevisiae : 3 S. pombe) RNA was extracted RiboPure Yeast Kit, Ambion, AM1926 Total RNA-Seq  and newly-synthesized RNA-seq libraries were generated from 1000 ng of total RNA using TruSeq Stranded Total RNA LT Sample Prep Kit with Ribo-Zero Gold (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, cytoplasmic and mitochondrial ribosomal RNA (rRNA) was removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the depleted RNA was fragmented into small pieces using divalent cations at 94oC for 2 minutes. Cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 10 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.
GEO Accession
GSM4377806