Comparative transcriptomic analysis of Staphylococcus epidermidis associated with periprosthetic joint infection under in vivo and in vitro conditions
Total RNA from in vivo and in vitro samples was isolated as previously described protocol (27). Briefly, cell suspensions were mechanically homogenized and genomic DNA and bacterial ribosomal RNA (rRNA) removed using the DNase I kit (QIAGEN, Germantown, MD, US) and the Ribo-Zero rRNA removal kit (Bacteria) (Illumina, San Diego, CA, US) protocols, respectively. Total sample RNA was isolated using the miRNeasy Serum/Plasma kit (QIAGEN). rRNA-depleted RNA was purified using the RNeasy MinElute Cleanup kit (QIAGEN) and eluted in a final volume of 30 mL RNase-free water. RNA quantity and quality were evaluated using a Qubit 2.0 Fluorometer coupled with a Qubit RNA high-sensitive assay kit (Thermo Fisher Scientific, Waltham, MA, US) and an Agilent 4200 TapeStation system (Agilent, Santa Clara, CA, US). A Ovation SoLo RNA-Seq System (NuGEN Technologies, San Carlos, CA, US) was used to prepare cDNA libraries, as previously described (32). External RNA Control Consortium RNA Spike-In Mix 1 (Thermo Fisher Scientific) was added to each total RNA sample to measure variability in the library generation process (33). Human rRNA sequences were depleted using the SoLo AnyDeplete probe mix (NuGEN Technologies). The resulting cDNA libraries were sequenced on an Illumina HiSeq 4000 platform with 10 samples multiplexed per lane, producing 100-nucleotide, paired-end reads.