## 4 Materials and Methods
### Validation of sRNA Candidates and Prediction of Verified sRNA Targets  
Eighteen candidates were randomly selected from those with read counts at least 100 and less than 100 respectively (Table 1). More sRNAs with read counts of at least 100 were selected because researchers usually have more interest in sRNAs of bacteria with high levels in sequencing results [32; 41]. They were verified by real-time quantitative PCR (qRT-PCR). Total RNA was obtained and purified as described previously. qRT-PCR for sRNAs was performed using a mi\(DETECT\) A TRACK(tm) qRT-PCR Kit (RiboBio, Guangzhou, China) according to the manufacturer's recommended protocol. The specific primers for sRNAs used in the qPCR assay were purchased from RiboBio. The _RNAhybrid_ algorithm was previously used for the prediction of target mRNAs in bacteria [42]. It is more suitable for target prediction of short-length RNA [43; 44]. The two most highly-expressedsRNAs were selected for target mRNA prediction using _RNAHybrid_ with a maximum of \(-20\) kcal/mol minimum free energy [45].  
Effect of Different Acid Stress Conditions on the Growth, Vitality, and Gene Expression of S. mutans  
To test whether acid can affect the growth and vitality of _S. mutans_ and the expression of sRNAs and their target mRNAs related to acid tolerance, we performed a previously described assay with minor modifications [20]. _S. mutans_ UA159 was incubated in 36 mL of BHI broth at 37 \({}^{\circ}\)C until the cell reached the stationary phase of growth. The cell cultures were then divided into 12 aliquots denoted 7.5-0.5, 7.5-1, 7.5-2, 6.5-0.5, 6.5-1, 6.5-2, 5.5-0.5, 5.5-1, 5.5-2, 4.5-10.5, 4.5-1, and 4.5-2. Subsequently, these 12 samples were centrifuged and resuspended in 3 mL of fresh BHI buffer at a pH of 7.5, 6.5, 5.5, or 4.5. The buffer used in a previous study was used as a reference [46]. After incubation at 37 \({}^{\circ}\)C for 0.5, 1, or 2 h, 200 \(\upmu\)L of each cell suspension was collected to measure the absorbance at 600 nm. For vitality assessment, 50 \(\upmu\)L of each cell suspension was mixed with 450 \(\upmu\)L of PBS. Bacterial samples (10 \(\upmu\)L) were inoculated by spiral plating onto BHI agar plates at three dilutions (\(10^{-6}\), \(10^{-7}\), and \(10^{-8}\)), and the total number of colony-forming units (CFUs) per volume (CFUs\(\cdot\)mL\({}^{-1}\)) were quantified after anaerobic incubation for 48 h at 37 \({}^{\circ}\)C. The cell suspensions remaining at each time point were harvested by centrifugation and used for RNA isolation. Total RNA was extracted, and qRT-PCR for sRNAs was performed as described previously. The target genes (\(glnQ\), \(glnM\), \(f\!f\!h\), \(brpA\), and \(relA\)) were reverse-transcribed using the PrimeScriptTM Reagent Kit (Takara, Dalian, China) according to the manufacturer's protocol. The qPCR reaction mixture (10 \(\upmu\)L) included 5 \(\upmu\)L of SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), 10 \(\upmu\)M of the specific forward and reverse primers (0.2 \(\upmu\)L for each), 1 \(\upmu\)L of the template cDNA, and 3.6 \(\upmu\)L of RNase-free water. The qPCR conditions included an initial denaturation at 95 \({}^{\circ}\)C for 20 s, followed by 40 cycles of denaturation at 95 \({}^{\circ}\)C for 10 s, annealing at 60 \({}^{\circ}\)C for 30 s, and extension at 70 \({}^{\circ}\)C for 1 s. The 16S rRNA levels under each condition were used for normalization. The specific forward and reverse primers for \(glnQ\), \(glnM\), \(f\!f\!h\), \(brpA\), and \(relA\) were described previously [14; 36; 37]. The primer sequences are shown in Table 2. The threshold cycle values (\(C_{\text{t}}\)) were determined, and the data were analyzed according to the \(2^{-\Delta\Delta\text{Ct}}\) method [47].