title: GSM6330129: Treat 75x rep 4; Danio rerio; RNA-Seq
library_construction_protocol: RNA was extracted from pools of 75 larvae using the RNA mini extraction kit (Qiagen, Hilden, Germany). Samples were lysed in RLT+ buffer with β-mercaptoethanol (Sigma-Aldrich, Overijse, Belgium) and homogenized 10 times with a 26-gauge needle in a 1 ml syringe. 22 ml of RNAse free water were used to resuspend total RNA. RNA extract was treated with DNAseI (Qiagen, Hilden, Germany) to avoid DNA contamination. The quantity (ng/ml) and quality of each extract was assessed by nanodrop spectrophotometer measurements (Thermofisher Scientific, Waltham, MA USA). Poor quality (260/280 < 2; 260/230 < 2) samples were subsequently purified by Lithium chloride precipitation, followed by 2 times pellet washing with 70% ethanol and resuspended in 40 µl of RNAse-free water and stored at -80°C. The integrity of total RNA extracts was assessed with BioAnalyzer analysis and provided RIN (RNA Integrity Number) scores for each sample (Agilent, Santa Clara, CA USA) cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit (Illumina, San Diego, CA USA) according to the manufacturer's instructions.