GSM3066093: PDBL10; Schizosaccharomyces pombe; RNA-Seq
Total RNA-seq libraries were prepared using TruSeq Stranded Total RNA with Ribo-Zero Gold rRNA Removal Kit (Yeast) (MRZY1324, Illumina, Paris, France). Briefly, starting with 2 microgrammes of total RNA, the first step involved the removal of cytoplasmic and mitochondrial ribosomal RNA (rRNA) using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the RNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. 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). Then surplus PCR primers were removed by purification using AMPure XP beads (Beckman Coulter). Final cDNA libraries were checked for quality and quantified using Fragment analyzer Automated CE System from Advanced Analytical Technologies, Inc.