RNA-seq of Lepomis macrochirus: adult muscle tissue
A 1.5 ľg sample of RNA was used to purify mRNA from the total RNA sample using poly-T oligo-attached magnetic beads. Following purification, the mRNA underwent random fragmentation in 5x NEBNext First Strand Synthesis Reaction Buffer. A double stranded cDNA library was then constructed according to the protocol set forth by the NEBNext Ultra RNA Library Prep Kit. Upon synthesis of the double stranded cDNA, adenylation of the 3 ends and ligation of adaptor sequences was performed prior to PCR enrichment. Library concentration was determined on a Qubit 2.0 fluorometer (Life Technologies) and quality was assessed with the Agilent Bioanalyzer 2100. Paired end reads were then generated on an Illumina Hi-Seq platform which produced a total of 84,129,866 raw reads.