RNAseq of Brochothrix thermosphacta in meat
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).