Total RNA was purified from bacterial cells as described above (Lasa et al. 2011 (link)). Ribosomal RNAs were removed from 10 µg of total RNA with the RiboZero Bacteria kit (Illumina, CA). Enriched mRNA was treated with E. coli Poly(A) Polymerase (NEB) in the presence of 10 mM ATP for 30 min at 37ºC to add a polyA tail. PolyAdenylated mRNAs were cleaned using the RNA Clean & Concentrator kit (Zymo, CA). Purified mRNAs were then converted into a nanopore-compatible RNA library using the Direct RNA Sequencing Kit from Oxford Nanopore (SQC-RNA002) (Pust et al. 2021 (link)). The library was sequenced on an R9.4 flowcell in a GridION instrument (Oxford Nanopore) for 24 h and base called in real-time in the instrument with the software Guppy. Data obtained by Oxford Nanopore are available at NCBI under Bioproject PRJNA922758 (S. aureus RN10359) and Bioproject PRJNA860339 (S. aureus MW2). The resulting reads were mapped using the Geneious Prime tool (Biomatters) using the S. aureus NCTC8325 genome as a reference (accession number: NC_007795.1) (Baba et al. 2008 (link)). The transcriptome mapping information is available on our website (https://staph.unavarra.es:10443).