Ribocop

Prior to RNA sequencing, total RNA samples were rRNA depleted using RiboCop (Lexogen) according to the manufacturer’s instructions. Samples were re-quantified using Qubit (Thermo Fisher Scientific), and up to 100 ng of RNA was used for library preparation using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB). The libraries were prepared according to the manufacturer’s instructions, quantified and quality checked using TapeStation (Agilent). The libraries were prepared with indexing primers and pooled into two library preps. There were eight chrRNA libraries that were pooled together and sequenced on a single G NEXTseq 500/550 (150) flow cell to obtain sequencing depth of approximately 40 M reads per sample. The 4 total RNA libraries were pooled with another 8 libraries not analyzed here; the 12 libraries were then loaded and sequenced on another G NEXTseq 500/550 (150) flow cell resulting in a lower sequencing depth. The RNA sequencing was performed at the Genomics Facility at the University of Birmingham. Briefly, the concentrations of the libraries were determined using Qubit and the average library size was determined using TapeStation (Agilent). The libraries were then diluted to 1.6 pM, and 1% of 20 pM PhiX control was added. The libraries were then loaded onto a flow cell and the sequencing was performed on Illumina NEXTseq.

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.