RNA-Seq of Nocardia cyriacigeorgica High Virulence Strains
Total RNA (1 g) was used for library construction with the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturers instructions. Poly(A)+ mRNA was enriched using Oligo(dT) magnetic beads and subsequently fragmented by divalent cations under elevated temperature. First-strand cDNA synthesis was performed using random oligonucleotide primers, followed by second-strand cDNA synthesis to generate double-stranded cDNA. The cDNA was purified, end-repaired, A-tailed at the 3 ends, and ligated to Illumina sequencing adapters. Libraries of approximately 400500 bp were size-selected using AMPure XP beads, PCR-amplified, and purified again with AMPure XP beads. Library quality was assessed using an Agilent 2100 Bioanalyzer with the High Sensitivity DNA Kit. Library concentration was measured using the PicoGreen dsDNA assay, and effective library concentration was determined by quantitative PCR. Equimolar amounts of multiplexed libraries were pooled, serially diluted, and sequenced on an Illumina platform in paired-end 150 bp (PE150) mode.