2025-07-18
ENA first public
ERC000011
ENA-CHECKLIST
MU
INSDC center name
public
INSDC status
ERS22652036
SRA accession
E-MTAB-14713:Exp-15-06-83_S153
Submitter Id
65
year
age
Blood culture: Streptococcus pneumoniae
blood culture
ArrayExpress
broker name
not collected
collection date
human
common name
Protocols: 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 spun down (3,200 x g, 10 min) and the supernatant was carefully removed. Cells were washed once with 1 ml NFW before pellets were dissolved in 100 μl NFW. 1 ml TRI reagentTM was added and samples were transferred into 2 ml bead-beating tubes containing 0.1 mm zirconia/silica beads (PureLink™ InvitrogenTM). Cells were lysed by 3x 1-min cycles of bead-beating at 3,000 rpm on a BioSpec Mini-Beadbeater-24 with 1 min breaks on ice. Samples were incubated at RT for 5 min before phase separation was performed by addition of 200 μl chloroform (MP Biomedical), followed by a 2 min incubation at RT and centrifugation at 12,000 x g for 15 min. RNA-containing aqueous phase was purified using the RNA Clean-and-Concentrator-25 kit (Zymo Research) according to the manufacturer’s instructions with a final elution volume of 30 μl. 40 U of RNaseOUT™ Recombinant Ribonuclease Inhibitor (40 U/μl, InvitrogenTM) were added to eluted RNA samples before any remaining DNA traces were removed using the Ambion® DNA-free™ DNase Treatment and Removal Reagents (Life Technologies) as per the manufacturer’s protocol. 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).
description
adult
developmental stage
Sepsis
disease
Wild type genotype
genotype
not collected
geographic location (country and/or sea)
TOPS study participant \83\
individual
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
blood
organism part
Homo sapiens
scientific_name
female
sex
Exp-15-06-83_S153
title