Bacteria protect reagent

The RNA content of exponential and/or stationary phase C. acnes cultures was extracted using the RNeasy® Mini Kit (Qiagen) complemented with Bacteria Protect Reagent (Qiagen), following the manufacturer’s instructions. Resulting RNA concentrations were measured using NanoDrop® (Saveen Werner) and samples stored in −20 °C until later use.
The relative abundance of roxP and gapdh transcripts was determined through quantitative real-time PCR (qPCR) using Power SYBR Green RNA-to-Ct 1-step Master Mix (ThermoFisher) mixed 1:1 (v/v) with 10 ng RNA and 100 nM primers (roxP: 5′-GCATCTAGCCCTCTCACCAT-3′ and 5′-CTGAGAGTCCGGTAGGTGGT-3′; gapdh: 5′-GCATCATGACTACCGTCCAC-3′ and 5′-CGGTGGTCTCCTTAGAGGTC-3′) in nuclease free water. Plates were run with reverse transcription for 30 min at 48 °C, 10 min at 95 °C, and 40 cycles of 15 sec at 95 °C alternating with 1 min at 60 °C, on an iCycler iQ (Bio-Rad). Results were interpreted via double delta Cq analysis, using the recorded values for C. acnes strain 266 as a reference. The experiment was run in biological and technological duplicates.

P. acnes mRNA was isolated using RNeasy Mini Kit (Qiagen) supplemented with Bacteria Protect Reagent (Qiagen). Briefly, 1 ml P. acnes culture was pelleted, resuspended in a mixture of PBS and Bacteria Protect Reagent and incubated for 5 min. Bacteria were lysed in TE-buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) containing 15 mg/ml lysozyme (Sigma-Aldrich) and proteinase K (Qiagen); incubating for 10 min with agitation. Reagent RLT (RNeasy kit) was added and particles removed by centrifugation. The remaining supernatant was mixed with ethanol, and the sample added to RNeasy spin columns according to manufacturer’s instructions.
Gene expression of roxP and gapdh was analyzed by quantitative real-time PCR (qPCR) using Power SYBR Green RNA-to-Ct 1-step Master Mix (ThermoFisher), 100 nM of primers (gapdh_for/rev, qRT-PCR_roxP_for/rev) (Table 1) together with 5–10 ng mRNA. Samples were run on an iCycler iQ (Bio-Rad) with reverse transcription at 48 °C for 30 min, followed by 95 °C for 10 min and 40 cycles with 15 s at 95 °C and 60 °C for 1 min. Gene expression levels were quantified using the ΔΔCt-method.

E. coli K12 DH5α transformed with puc18 plasmid (Sigma-Aldrich-Germany) were cultured in Luria Bertani (LB) medium with 100 μg/ml ampicillin (Sigma-Aldrich-Germany) under aerobic conditions. Overnight cultures were inoculated at a dilution of 1:100 into a new LB medium with ampicillin and were incubated at 37 °C with shaking at 200 rpm until they reached mid-exponential phase (OD600 = 0.6). Bacteria protect reagent (Qiagen-Germany) was added and the bacterial cultures were centrifuged at 4000 rpm for 10 min. The bacterial pellets were collected for extraction of total RNA followed by next generation sequencing and quantitative real-time PCR (qRT-PCR) assays.