Comparative cycle threshold method

For RNA stabilization, the sampled culture was mixed with an equal volume of RNAprotect bacterial reagent (Qiagen). After incubation at room temperature, cells were collected by centrifugation and stored at −80 °C. Total RNA was isolated using NucleoSpin^® RNA (MACHEREY-NAGEL, Düren, Germany) according to the manufacturer’s instructions. Purified RNA was further treated with DNaseI (Takara Bio Inc., Shiga, Japan), ethanol precipitated, and suspended in RNase-free water as described previously [35 (link)].
qRT-PCR was performed using the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and Power SYBR^® Green PCR Master Mix with MuLV Reverse Transcriptase and RNase Inhibitor from the GeneAmp RNA PCR Kit (Thermo Fisher Scientific) as described previously [17 (link)]. The primers listed in Table S2 were used. The comparative threshold cycle method (Thermo Fisher Scientific) was used to quantify relative expression, and the relative expression ratios of each gene were normalized using the values for 16S rRNA.

qRT-PCR was performed using the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and Power SYBR^® Green PCR Master Mix with MuLV Reverse Transcriptase and RNase Inhibitor from the GeneAmp RNA PCR Kit (Thermo Fisher Scientific), as previously described [26 (link)]. The primers used are listed in Table S1. The comparative threshold cycle method (Thermo Fisher Scientific) was used to quantify the relative expression, and the relative expression ratios of each gene were normalized using the values for 16S rRNA.

S. aureus strains were grown in Luria-Bertani medium and then harvested. Cells were isolated by centrifugation and dissolved in a solution of 33 mmol/L sodium acetate, 17 mmol/L sodium dodecyl sulfate, and 1 mmol/L EDTA (pH 5.5). The cells were then mixed with glass beads and lysed by using a Fast Prep Apparatus (MP Biochemicals, LLC, Santa Ana, CA, USA).
RNAs were isolated by using water-saturated phenol (pH 5.0). RNAs were precipitated and washed with ethanol. Northern blotting of RNA markers was conducted by loading 10 μg of total RNA onto 8 mol/L urea, 8% polyacrylamide gels. Gels were subjected to electrophoresis and blotted onto nylon membranes at 30 V for 1.5 h in 0.5× Tris-HCl, borate, EDTA buffer.
Prehybridization and hybridization were performed by using ExpressHyb solution (Clontech, Mountain View, CA, USA) and ³²P-labeled DNAs (Technical Appendix Table 2). Signals were detected by using phosphorimaging (Biocompare, South San Francisco, CA, USA) and quantified. Expression levels of sRNAs in strains were monitored by using quantitative PCR and specific primers (Technical Appendix Table 3). cDNAs were produced by using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Using the comparative cycle threshold method (Applied Biosystems), we normalized sRNA counts against transfer–messenger RNA (tmRNA) and S. aureus reference strain L102.