GSM9669533: Staphylococcus lugdunensis ATCC 43809 grown in TSB containing C. acnes CFCM 2X, replicate 1; Staphylococcus lugdunensis; RNA-Seq
After incubation, RNA protect Bacteria Reagent (Qiagen, Germantown, MD, USA) was added, as per the manufacturer's recommendations. The culture + RNA protect was homogenized for 5 seconds using a vortex and kept at room temperature for 5 minutes. The mixture was then centrifuged for 10 minutes (12000 x g) and the supernatant discarded. The pellets were air dried, and a lysis solution containing 20 mg/mL lysozyme (MIDSCI, Fenton, MO, USA), 40 μg/mL lysostaphin (Sigma- Aldrich), and 4 mg/mL proteinase K (Sigma-Aldrich) was added followed by an incubation step at 37 °C for 1 h (homogenization was done using a vortex for 10 seconds every 10 minutes). Purification was performed using the Promega SV Total RNA Isolation System following the manufacturer's instructions. To remove contaminating DNA, total RNA was treated with TURBO DNase (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions, followed by an additional purification step using the SV Total RNA Isolation System (Promega), according to the manufacturer's recommendations. RNA concentration and purity were assessed using an ND-1000 NanoDrop 246 spectrophotometer (Thermo Fisher Scientific). Absence of DNA contamination in the RNA was checked by quantitative Real-Time PCR. RNA sequencing (RNA-seq) was performed on an Illumina NextSeq 2000 platform using a P1 flow cell, as single reads (100 bp) with sequencing depth estimated to be about 4.4x. at The University of Kansas Genome Sequencing Core.