title: GSM5915256: Trunk treatment-rod rep3; Micropterus salmoides; RNA-Seq
library_construction_protocol: Total RNA was isolated from males only using 30-50 mg of the frozen trunk and head kidney tissue using Stat-60 (Tel-test Friendswood, TX, USA) reagent. Each tissue was homogenized in 700 µl Stat-60, 150 µl chloroform was added and the samples were centrifuged at 20,800 x g for 15 min at 4ºC. This extraction was repeated with fresh STAT-60, and the RNA was precipitated overnight at -20 ºC in isopropanol with the addition of 1.5 µL of GlycoBlueTM (Ambion, Invitrogen, Austin, TX) as a coprecipitant. Following centrifugation at 20,800 x g for 45 minutes, the pellet was washed twice with 70% ethanol, air-dried, and resuspended in 20 mL of RNA secure and heated up at 60ºC for 10 minutes (Ambion, Austin, TX, USA). We performed DNase treatment to remove possible contamination with genomic DNA using Turbo DNA-freeTM kit (Ambion, Austin, TX, USA). The RNA-seq library was performed by using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, catalog # E7490) and the NEBNext Ultra II  Directional RNA Library Prep Kit (New England Biolabs, catalog #E7760) according to the manufacturer's user guide. Briefly, 250 ng of total RNA was used for Poly(A) selection, then  fragmented in NEBNext First Strand Synthesis Buffer via incubation at 94 °C for 10min. The first-strand cDNA synthesis using reverse transcriptase and random hexamer primer.  Synthesis of ds-cDNA was performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation.  At this point, Illumina adaptors were ligated to the sample.  Finally, each library (uniquely barcoded) was enriched by 10 cycles of amplification, and purified with Agencourt AMPure beads (Beckman Coulter, catalog # A63881). The barcoded libraries were sized on the Bioanalyzer and quantified with the Qubit® 2.0 Fluorometer. Finally, individual libraries were pooled in equimolar concentration.