Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: The organs of the fishes were dissected and transferred into 2 ml tubes with 1 ml cooled QIAzol (Qiagen, Hilden, Germany) and one 5 mm stainless steel bead (Qiagen) was added. Homogenization was performed using a TissueLyzer II (Qiagen) at 30 Hz for 3x 1 min. After incubation for 5 min at room temperature 200 µl chloroform was added. The tube was shaken for 15 s and incubated for 3 min at room temperature. Phase separation was achieved by centrifugation at 12,000x g for 20 min at 4°C. The aqueous phase was transferred into a fresh cup and 10 µg of Glycogen (Invitrogen, Darmstadt, Germany), 0.16x volume NaAc (2 M; pH 4.0) and 1.1x volume isopropanol were added, mixed thoroughly and incubated for 10 min at room temperature. The RNA was precipitated by a centrifugation step with 12,000 x g at 4°C for 20 min. The supernatant was removed and the pellet was washed with 80% Ethanol twice and air dried for 10 min. The RNA was resuspended in 20 µl DEPC-treated water by pipetting up and down, followed by incubation at 65°C for 5 min. The RNA was quantified with a NanoDrop 1000 (PeqLab, Erlangen, Germany) and stored at -80°C until use. Sequencing procedure was done using Illumina methodology [Bentley et al, 2008]. Around 2.5 µg of total RNA was used for library preparation (Illumina, TruSeq™ RNA Sample Prep Kit v2) using the manufacturer’s description.