550 platform

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.

Clinical samples were collected by following the standards of aseptic processing procedures: (1) A 1.5–3 mL of the BALF sample or 5 mL of blood sample was collected from each patient. Nucleic acid extraction took place using a TIANamp Micro DNA Kit (DP316, Tiangen Biotech Co., Beijing, China). (2) DNA libraries were constructed using the VAHTS Universal Plus DNA Library Prep Kit for Illumina (ND617-C2, Vazyme Biotech Co., Nanjing, China). Agilent 2100 was used for quality control of the DNA libraries. Qualified libraries were sequenced by the Illumina 550 platform. (3) High-quality sequencing data were generated, followed by computational subtraction of human host sequences mapping to the human reference genome (hg19) using Kraken2 2.1.2 and Burrows-Wheeler Alignment (BWA). Low-quality and short reads (length < 50 bp) were removed by fastp 0.20.1. The remaining data were aligned to the Pathogenic Microbial Genome Databases, including bacteria, viruses, fungi, and parasites. The classification reference databases were downloaded from National Center of Biotechnology Information (NCBI) (ftp://ftp.ncbi.nlm.nih.gov/genomes/) and the mapped data were processed for advanced analysis.