Cells were mixed in a defined ratio (1 S. cerevisiae : 3 S. pombe) RNA was extracted RiboPure Yeast Kit, Ambion, AM1926 Total RNA-Seq libraries were generated from 800 (Total RNA ) or 300 (newly-synthesized RNA) ng of RNA using TruSeq Stranded Total RNA Library Prep Gold kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, CA) with modifications. Briefly, ribosomal RNA was removed using biotinylated target-specific oligos, riboPOOL targeting S. cerevisiae ribosomal RNA (siTOOLs Biotech, Planegg/Martinsried, DE) combined with Dynabeads MyOne Streptavidin C1 (Thermo Fisher) according to manufacturer's instructions. Following purification on Agencourt RNAClean XP (Beckman-Coulter, Villepinte, France) with a 1.8X ratio, 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 12 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
GSM4377775