GSM5971569: Fish 24; Salmo salar; RNA-Seq
Homogenization of liver tissue was conducted with a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was extracted and treated with DNase using the BioRobot EZ1 and RNA Tissue Mini Kit (Qiagen, Hilden, Germany). Characterization of total RNA was done with the Agilent 2100 Bioanalyzer using the 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA, USA). The RNA integrity number (RIN), which is a measure of degradation in RNA samples, was 9.5 ± 0.2 (n=40) in liver (mean ± st.dev). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions.