Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Each fraction was deproteinized by addition of 1% SDS and 1 volume of hot phenol and shaking at 1,500 rpm at 55°C for 5 min. Phases were separated by centrifugation at 12,000 g for 15 min at 4°C. The aqueous phases were added glycogen to 50 µg/ml and RNA was precipitated with 60% isopropanol. The RNA pellets were dissolved in 35 µl of DEPC-treated sterile MilliQ water and stored at -80°C. Each fraction sample was added 85 pg/µl of the spike-in RNA (5’P-CUCGUCCGACGUCACCUAGA, IBA). RNA-seq libraries were prepared by Vertis AG (Freising-Weihenstephan, Germany). Briefly, RNA was polyadenylated with poly(A) polymerase, 5’-triphosphates were removed with tobacco acid pyrophosphatase followed by ligation of a 5’-adapter. First-strand cDNA synthesis was performed with the use of an oligo(dT) barcoded adapter primer and the M-MVL reverse transcriptase. The resulting cDNA was PCR-amplified with a high fidelity DNA polymerase. cDNA was purified with the Agencourt AMPure XP kit (Beckman Coulter Genomics).