## 2 Materials and Methods
### Effects of MLH on Gene Expression in MRSA  
For RNA extraction, MRSA cells were harvested at 0, 1, and 5 h after treatment, with samples at 5, 6, and 12 h of the growth curve. The ODs at 600 nm were adjusted to 0.5 for normalization. TRIzol(r) (0.75 mL) reagent was added to 0.25 mL of each sample to form a pellet. The samples were mixed vigorously and then centrifuged at 12,000\(\times\)\(g\) at 4 \({}^{\circ}\)C for 5 min. The clear supernatant was then transferred to a new tube. Chloroform (0.2 mL) was added to 1 mL of TRIzol(r) Reagent for lysis [15] and the tube was securely capped. The samples were incubated at \(-\)80 \({}^{\circ}\)C for 1 h, then centrifuged at 12,000\(\times\)\(g\) for 15 min at 4 \({}^{\circ}\)C. The mixture separated into three phases: a lower red phenol-chloroform, an interphase, and a colorless aqueous phase. The RNA contained in the aqueous phase was moved to a new tube. The pellet was resuspended in 1 mL of 75% ethanol per 1 mL of TRIzol(r) Reagent for lysis. The sample was vortexed briefly, then centrifuged for 5 min at 7500\(\times\)\(g\) at 4 \({}^{\circ}\)C. The supernatant was discarded. The RNA pellets were placed into a vacuum for 5-10 min. DNase I was used to prevent DNA contamination. The purity and concentration of RNA was confirmed by a Thermo Fisher Scientific(tm) NanoDrop 2000 and Qubit(tm) RNA Assay Kits with an Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA) [16].  
RNA sequencing was performed at Prince of Songkla University, Thailand. The Ribo-Zero(tm) Magnetic Kit (Epicentre Biotechnologies, Madison, WI, USA) was used to deplete non-coding RNAs from the samples. Then, the Epicentre ScriptSeq v2 RNA-Seq Library Preparation Kit (Catalogue No. 20021533) was used to prepare RNA-seq libraries that were started with 20-25 ng of enriched RNA. After 13 cycles of amplification, AMPure XP beads were used to purify the RNA-seq libraries. Finally, RNA-seq libraries were unified in equimolar amounts and the quantity and quality of each unit were adjudicated by a Bioanalyzer. The resultant pools were subsequently sequenced on an Illumina NextSeq 550 system that yielded a total of 25,783,620 paired reads. The quality trimmed FASTQ files were used to calculate the total number of paired reads, the average Phred quality scores, and the average GC content [17].  
The standard method for measuring and comparing levels of gene expression is high-throughput sequencing of messenger RNA (RNA-seq). RNA-seq experiments reduce the raw read data to comprehensible results that generate very large, complex data sets using software. The free open-source software tools HISAT, StringTie, and Ballgown were used for comprehensive analysis of RNA-seq experiments. Paired-end 250 bp reads were aligned with the _S. aureus_ subsp. _aureus_ NCTC 8325 (accession no. NC_007795.1) using HISAT2 (v. 2.1.0) and then we quantified gene abundance using StringTie (v. 2.1.1) [17]. Finally, statistical analysis of differential gene expression was conducted using R software (v. 3.0.1) with the Ballgown package (v. 2.2.0) [17]. Differential gene expression was defined as such when the false discovery rate was \(\leq\)0.05 and differential expression \(\geq\)2.