Total rna isolation kit v2

Total RNA was extracted from S. aureus RN450 and graS^FS strains using Total RNA Isolation Kit V2 (Vazyme, Nanjing, Jiangsu, China, cat.# RC112-01) according to the manufacturer’s instructions. Reverse transcription was performed with Hifair^® III 1st Strand cDNA Synthesis SuperMix (Yeasen, Shanghai, China, cat.# 11141ES10). Quantitative PCR was performed with SYBR green master mix (Yeasen, Shanghai, China, cat.# 11201ES03). Standard curve was generated by serially diluting the pOS1-mprF or pOS1- dlt plasmids (10¹-10⁸ copies) and determining the corresponding cycle threshold. According to the standard curves, the mRNA copies of each sample were calculated and normalized to internal control (16S rRNA) of each S. aureus sample before mRNA abundance from different strains treated under the same condition or from the same stain undergoing different treatment was compared.

For RNA extraction, ground lung tissue and BEAS-2B were utilized. Total RNA was extracted using the Total RNA Isolation Kit V2 (Vazyme, China), and cDNA was produced using the HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme, China). RT-qPCR was performed on an ABI 7500 thermocycler (Applied Biosystems, Foster City, CA, USA). The mRNA expression levels of GPX4 and ACSL4 were assessed using the SYBR Green PCR Master Mix (Vazyme). The suitable primers were summarized in Table 1. The relative expression levels of mRNA were reported as fold change compared to levels detected in controls via the ΔΔCt method.Table 1

PCR primer sequence

Genes	Primer sequence
Hsa-GPX4	F:CCGCTGTGGAAGTGGATGAAGATC
	R: CTTGTCGATGAGGAACTGTGGAG
Hsa-ACSL4	F: TCTGCTTCTGCTGCCCAATT
Has-IL-6 Has-TNFα	R: CGCCTTCTTGCCAGTCTTTT F: GGTGTTGCCTGCTGCCTTCC R: GTTCTGAAGAGGTGAGTGGCTGTC F: TGGCGTGGAGCTGAGAGATAACC R: CGATGCGGCTGATGGTGTGG
Hsa-β-ACTIN	F: TGGCACCCAGCACAATGAA
	R: CTAAGTCATAGTCCGCCTAGAAGCA
Rat-GPX4	F: GGCAGGAGCCAGGAAGTAAT
	R: TGGGCATCGTCCCCATTTAC
Rat-ACSL4	F: GACAGAATCATGCGGTGCTG
	R: TAACCACCTTCCTGCCAGTC
Rat-β-ACTIN	F: CGCGAGTACAACCTTCTTGC
	R: CCTTCTGACCCATACCCACC

RNA was extracted from macrophages or tumor cells using Total RNA Isolation Kit V2 (Vazyme, Cat# RC112-01). Reverse transcription from RNA to cDNA use Hiscript Reverse Transcriptase (Vazyme, Cat# R302-01). PCR reactions were performed on a CFX96 Real-Time PCR System (Bio-Rad Laboratories) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Cat# Q711-02). All the primers for qRT-PCR used in this study were shown in Supplementary Table 2.