Dual-species RNA-seq of P. aeruginosa PA14 and S. aureus USA300 JE2 coculture
RNA was extracted as previously described (Lee et al. mBio 2025). Briefly RNA from cell pellets were extracted into organic reagent using physical lysis and purified using a Qiagen RNeasy kit. Following DNAseI treatment and  precipitation, RNA samples were submitted to the MIT BioMicroCenter for further sample preparation. Sample integrity was validated using fluorescence-based electrophoresis (AATI Fragment Analyzer), following which ribosomal depletion was performed using the NEB Next rRNA Depletion Kit (New England Biolabs). Subsequently, library preparation (adapter ligation, size selection, barcoding, enrichment) was performed using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). The quality of the resulting libraries was validated using real-time PCR, following which the libraries were pooled and sequenced on a single lane of an Ilumina NextSeq500 instrument using 75 nt chemistry.