title: GSM3043298: flc1_2_F3_m1g; Arapaima gigas; RNA-Seq
library_construction_protocol: Total RNA was isolated from gonads and brain tissue of six A. gigas individuals using the TRIzol Reagent (Applied Biosystems, USA) as per manufacturer's protocol. Total RNA was treated for 10 min at 65 °C with RNAsecure reagent (Ambion, USA). To eliminate genomic DNA contamination, all RNA preparations were treated with RNase-free Turbo DNase (Ambion, USA) for 15 min at 37 °C. Total RNA was further purified using RNAeasy Mini RNA kit (Qiagen) as per manufacturer's protocol. For the creation of cDNA libraries, poly(A) mRNA was isolated by two consecutive purification cycles on oligo d(T) cellulose using a Micro-PolyA-Purist kit (Ambion, USA). RNA was quantified using NanoDrop® 2000 spectrophotometer (Thermo Scientific, USA) and the quality checked on an agarose gel (electrophoresis in 1.5% w/v) and Bioanalyzer 2100 (Agilent Inc., USA) using the RNA 6000 Nano Kit (Agilent Inc., USA). For quality control, two aliquots of RNA were taken per extraction, one was left at 37 °C for two hours, while the other was stored at 4 °C. Both aliquots were run on an Agilent RNA nano-chip (Agilent Inc., USA) and the resulting electropherograms inspected for signs of degradation. The samples of RNA total had an RNA Integrity Number (RIN) value between 8.0-8.5. Equal volumes of RNA from each sample of tissue were used for cDNA synthesis. Approximately 500 ng of mRNA from each tissues sample was used to construct RNA-Seq libraries with the SOLiD™ Total RNA-Seq Kit for Whole Transcriptome Libraries (Applied Biosystems, USA) following the manufacturer's instructions. Barcodes were used to identify each library individually. A total of 24 sequencing libraries were constructed. Each sample tissue of the gonads and brain (three male and three female) was sequenced individually as biological replicates. The libraries were deposited on six lanes of a slide and sequenced using a SOLiD 4 sequencer (Applied Biosystems, USA) to generate 50nt short sequencing read. Sequencing was performed in the “Laboratório de Ecofisiologia e Evolução do Instituto Nacional de Pesquisas da Amazônia”, Manaus, Brazil.