GSM5417082: S epidermidis SECOM005A; Staphylococcus epidermidis; RNA-Seq
Total RNA was extracted using RNeasy mini kit (QIAGEN) as described in França et al., 2012, PLoS One 7 (5): e37480. In brief, cells were suspended in 500 µL of the lysis buffer provided by the kit plus 500 µL of phenol. This suspension was transferred into BeadBug tubes (1.0 mm Zirconium beads, triple-pure, high impact, Sigma) and using an MPBio 24 (MPBio) cells disruptor, the cells were lysed (3× 6.5m/s for 35 seconds, with 5 minutes intervals on ice between cycles). Finally, the tubes were centrifuged, and the suspension transferred into a new tube. An equal volume of 70% of ethanol was added, the suspension transferred into the RNeasy mini kit columns and the manufacturer's instructions were strictly followed. RNA quality was determined using Experion™ Automated Electrophoresis System and RNA quality indicators were above 9 for all samples. Thereafter, total RNA obtained from three independent experiments were mixed and treated with TURBO DNase (Ambion) and acid-phenol:chloroform: isoamyl alcohol (125:24:1) (Ambion, MA, USA) to degrade and isolate, respectively, genomic DNA. Additionally, total RNA was treated with MICROBEnrichTM (Ambion) to remove contaminating eukaryotic RNA. Finally, before the library construction, a Ribo-ZeroTM rRNA removal kit for Gram-positive bacteria (Epicentre) was used to remove bacterial ribosomal RNA. Libraries were constructed using ScriptSeq RNAseq Library preparation kit (low input) (Epicentre) and strictly following the manufacturer's indications. The quality of the libraries constructed was assessed by quantitative PCR and Hi-Sensitivity D1K TapeStation (Agilent 2200 TapeStation). Finally, all libraries were multiplexed, and sequencing data were generated in a MiSeq® sequencer (Illumina) from paired-end reads (2 × 150 bp).