title: Characterising Commensal and Pathogenic Staphylococcal Interactions with Neonatal and Adult Blood
design: Characterising Commensal and Pathogenic Staphylococcal Interactions with Neonatal and Adult Blood
library_construction_protocol: Hospitalised adult patients with a suspicion of sepsis referred for ICU admission at Fiona Stanley Hospital in Perth, Western Australia, were recruited to an ongoing prospective cohort sepsis study (The One Project Study, TOPS). Peripheral blood samples were collected from study participants by venepuncture into BACTEC™ Peds Plus™ (BD, 40 ml) and EDTA tubes (BD Vacutainer®, 10 ml). Within 2 hours of blood sample collection, EDTA blood samples were aliquoted for transcriptional (0.5 ml), genomic (4 ml), and plasma metabolomic (5.5 ml) analyses. For this study, 0.5 ml blood samples were stabilised into pre-aliquoted PAXgeneTM Blood RNA solution (1.38 ml) and stored at -80°C. Routine blood culture (BC) analysis was and relevant data (BC result, culture conditions, causative pathogen, BC time-to-positivity, sex) were obtained. Blood samples collected as part of the clinical trial were collected as close as possible to the routine blood culture sample. A subset (n=8) of BC-positive samples, collected between September 2021 and January 2023, were selected for dual RNA-sequencing based on the relevance of causative pathogen and BC time-to-positivity. Host and bacterial RNA was purified from sepsis samples as described above using our previously optimised protocol. Total RNA was quantified, and RNA quality was assessed as previously described. A modified TRI reagentTM (Sigma) protocol was used to extract total RNA from PAXgene™-stabilised blood samples. Briefly, samples (1.88 ml) were Host and bacterial rRNA was depleted from purified RNA samples using the Illumina Ribo-Zero Plus RNA Depletion kit (Illumina) following the manufacturer's guidelines. The efficacy of dual species rRNA depletion was verified using TapeStation. For cDNA library preparation, an in-house adapted SmartSeq assay protocol [42] using oligoT and random hexamer primers was employed. Full-length cDNAs were amplified, purified, and quantified using the Promega Quantus™ Fluorometer. Double-stranded cDNA was prepared for sequencing using the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina (New England Biolabs).