RNAseq of  Brochothrix thermosphacta in shrimp
RNA-Seq libraries  were constructed with the Truseq stranded mRNA sample preparation (Low  throughput protocol) kit from Illumina. Purified mRNA (10 ng) were cleaved  into small fragments using divalent cations under elevated temperature. The  cleaved RNA fragments were converted into a first strand cDNA using  SuperScript II reverse transcriptase (Invitrogen), actinomycin D and random  hexamer primers. Second strand cDNA was synthesized by replacing dTTP with  dUTP. These cDNA fragments then have the addition of a single 'A' base and  subsequent ligation of the adapter. The products were then purified and  enriched with 15 cycles of PCR. The final cDNA libraries were validated with  a Fragment Analyzer (Advanced Analytical, Ankeny, IA) and quantified with a  KAPA qPCR kit (Kapa Biosystems, Wilmington, MA).