Multispecies RNA Extraction and Sequencing

The RNA extraction was performed according to a previously described protocol (Bastakoti et al., 2023 (link)) following the recommendations from the manufacturer (RNeasy Mini Kit, Cat. No. 74104). Briefly, all samples were lysed enzymatically using lysozyme and lysostaphin, as well as mechanically disrupted using a homogenizer (Precellys Evolution, Bertin technologies) before RNA extraction followed by DNase treatment. Total RNA extracted from three replicates of S. aureus co-cultured with S. anginosus in the absence/presence of host cells collected at the time point of 1 h and 3 h, were processed for RNA-seq library preparation, as described previously (Bastakoti et al., 2023 (link)), using Lexogen’s CORALL™ Total RNA-Seq Kit with RiboCop (Cat.No.96; EU, CH, USA). No prior RNA fragmentation was needed in this protocol. The samples were sequenced on an Illumina 550 platform, with dual indexes, and paired end (PE) mode. The final sequencing concentration was 1.8 pM. The expected fragment length for PE reads was < 100 nucleotides.