RNA-seq of Brook Trout Liver
Total RNA was isolated using standard methods.  RNA was polyA selected to enrich for mRNA, then fragmented chemically.  After the mRNA was fragmented, cDNA was synthesized from the mRNA by reverse transcription (RT).  Platform-specific RT primers, designed with a random 3-nucleotide barcode, anneal to the fragmented mRNA and extend to the 5  end of the mRNA.  The SMARTScribe RT enzyme inserts a non-templated  CCC  sequence to the 3  end of the first synthesized strand.  This allows the SMART7.5 strand-switching primer s  GGG  sequence to anneal to the  CCC  sequence, which allows extension of the template for the first synthesized cDNA strand to include PCR primer sequences. AMPure bead purification was used to separate the primers from the cDNA.   The cDNA was subsequently amplified for a small number of cycles by PCR and products of the desired size range (400-500 bp) purified on agarose gels.