7500 sequence detector

Cells were grown as described above in 1.5 mL TSB, harvested and broken with a Mini-Beadbeater (Biospec Products) at maximum speed for 30 s. After incubation on ice for 5 min, the samples were centrifuged; and the supernatant was used to isolate total RNA according to the manufacturer’s instructions (Qiagen). After DNase treatment with a TURBO DNA-free TM kit (Ambion), approximately 2 μg of total RNA were reverse-transcribed with a PrimeScript RT reagent kit (Qiagen). The cDNA was used as a template for real-time PCR using SYBR-green PCR reagents (Roche). Reactions were performed in a MicroAmp Optical 96-well reaction plate using a 7500 Sequence Detector (Applied Biosystems). Primers used are listed in Supplementary Table 8. All RT-PCR experiments were performed in triplicate with gyrB as an internal control. All experiments were repeated independently at least three times with similar results.

Total RNA was isolated from cells by using TRI Reagent (Sigma, Milan, Italy). The amount and purity of RNA were determined spectrophotometrically. cDNAwas obtained by incubating 2 μg of total RNA with 4 U/μL of M-MLV reverse transcriptase (Promega, San Luis Obispo, CA, USA) according to the manufacturer’s instructions. Quantitative real time PCR (qPCR) was performed as reported in [51 (link)] using the GoTaq^® Probe Systems (Promega). The qPCR analysis was carried out in triplicate using an Applied Biosystems 7500 Sequence Detector with the default PCR setting: 40 cycles at 95 °C for 15 s, 60 °C for 60 s. mRNA was quantified with the DDCt method as described [52 (link)]. mRNA levels were normalized to β2 microglobulin as an endogenous control. The primer sequences used are listed in Table 1.

The S. epidermidis strains were grown to stationary phase. The cells were centrifuged and the pellets were lysed with a FastPrep-96 (MP Biomedicals Products) at 800 rpm for 300 s three times. Supernatants were collected for total RNA isolation according to the manufacturer’s instructions (Qiagen). After DNase treatment with a TRUBO DNA-free kit (Ambion), 1 μg of total RNA was reverse transcribed with a Prime Script RT reagent kit (Qiagen). The cDNA was used as a template for RT-PCR with SYBR Green PCR reagents (Roche). Reactions were performed in a MicroAmp optical 96-well reaction plate with a 7500 sequence detector (Applied Biosystems).

First-strand cDNA was generated using the SuperScript III First-Strand Synthesis System for RT-PCR Kit (Invitrogen), with random hexamers, according to the manufacturer’s protocol. PCR was performed in a total volume of 25 µl containing 1× Taqman Universal PCR Master mix, no AmpErase UNG, and 2.5 µl of cDNA; pre-designed, gene-specific primers, and probe sets for each gene were obtained from Assay-on-Demande Gene Expression Products (Applied Biosystems).
Real-time PCR reactions were carried out in a two-tube system and in singleplex. The real-time amplifications included 10 min at 95°C (AmpliTaq Gold activation), followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min. Thermocycling and signal detection were performed with 7500 Sequence Detector (Applied Biosystems). Signals were detected according to the manufacturer’s instructions and the relative expression levels were calculated as it has been previously described (9 (link)).

The nine genes (APC, FOXA1, MGMT, RARβ2, RASSF1A, SCGB3A1, SEPT9, SHOX2 and SOX17) promoter methylation levels were assessed by multiplex qMSP, using amplified DNA as template [25 (link),55 (link)]. Primers and probes specifically designed for the modified gene sequence plus the fluorochromes and quenchers used for each probe are listed in Supplementary Table S6. β-Actin was used as reference gene to normalize the DNA quantity of each sample [18 (link)]. 6 µL of WGA amplified DNA and Xpert Fast Probe (GRiSP, Porto, Portugal) were used in each multiplex qMSP reaction. Multiplex qMSP assays were carried out in 96-well plates in triplicate using a 7500 Sequence Detector (Applied Biosystems, Perkin Elmer, CA, USA). Sterile distilled water subjected to WGA was used a negative control and included in all plates. WGA amplified CpGenome™ Universal Methylated DNA (Merck Millipore, Burlington, MA, USA) subjected to six serial dilutions (5× factor dilution) was used to generate a standard curve in each plate, allowing for relative quantification and PCR efficiency evaluation. Efficiency values above 90% were considered. Relative methylation levels were determined as the ratio between the mean methylation levels of each gene and the respective value for β-Actin (the housekeeping gene), multiplied by 1000 for easier tabulation.

The expression of adhesion and biofilm formation genes in ST188 was detected by RT-PCR. We chose ST398 as the reference group because ST398 is an important host-species-adaptable S. aureus lineage with high virulence but low biofilm formation ability^{18 (link),21 (link)}. Complementary DNA was synthesized from total RNA using the QuantiTect reverse transcription system (Qiagen) according to the manufacturer’s instructions. Complementary DNA samples were amplified using the QuantiTect SYBR green PCR kit (Qiagen). Reactions were performed using a 7500 Sequence Detector (Applied Biosystems). We used purified chromosomal DNA at concentrations of 0.005–50 ng/ml to form a standard curve. All quantitative reverse-transcription polymerase chain reaction (qRT-PCR) experiments were performed in duplicate with gyrB as a control.