Non-normalised cDNA from Oncorhynchus mykiss MOM brain
Oncorhynchus mykiss
100 MOM (monosex males) of 47 daf (days after fertilization) rainbow trouts were anesthetized in ice. Brains were rapidly removed and immediately frozen in liquid nitrogen, then stored at -80°C. Total RNA was extracted using TRizol (Invitrogen, Cergy-Pontoise, France) according to the manufacturer's instructions. Total RNA yield was estimated by using a Nanodrop 1000 spectrophotometer (Labtech, Palaiseau, France) and RNA quality was checked with an Agilent Bioanalyzer (Agilent Technologies, Massy, France). Only good quality total RNA (RNA Integrity Number (RIN)&gt;8) were selected. Total RNA from several extractions were pooled to minimize the impact of biological variance and to have a large quantity necessary to the library preparation.   mRNA were then isolated from 250µg of total RNA using the Nucleotrap® mRNA kit (Macherey-Nagel, Hoerdt, France) as described in the manufacturer's manual, except for the elution procedure (40µl prewarmed (68°C) H2O for 40µl Oligo(dT) latex beads used, 3 rounds of elution). Quality of mRNA and depletion in ribosomal RNA were checked with the Agilent Bioanalyzer (Agilent Technologies, Massy, France). Different isolations of mRNA were made per sample, pooled and then ethanol precipitated overnight (2 vol of cold absolute ETOH, 1/10 vol of 3M pH5.2 Sodium Acetate and 20µg of glycogen). Double stranded cDNA were prepared as described by Leroux et al. [2010] using a modified protocol of the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Cergy-Pontoise, France). Briefly, a modified primer (5' GAGAGAGAGACTGGAG(T)16VN 3') containing the GsuI restriction site and methylated dCTP (10mM dm5CTP, Fermentas, Saint-Remy-les-Chevreuse, France) were used during the first strand cDNA synthesis. First strand was synthesized from 3µg of mRNA. Next steps (second strand synthesis, RNase I digestion, phenol/chloroform extraction and ethanol precipitation) were proceeded according to the manufacturer's instructions. Pellets were resuspended in 9µl of water. 3 to 5 independent synthesis were made for each sample and pooled in order to obtain a total of 3.9µg. Then, the double-stranded cDNA was digested by GsuI (5U) to remove the polyA tail for 90min at 30°C. cDNAs were quantified using Quant-iT PicoGreen kit (Invitrogen, Cergy-Pontoise, France).