RNA-Seq Analysis to investigate the impact of hypoxia on the host responses to Nontypeable Haemophilus influenzae infection in the human explanted lung

Hypoxia is a common microenvironmental condition in the mucus-obstructed, infected, and inflamed airways of lung disease patients and is known to influence immune responses in the lungs. However, the effects of hypoxia on lung host defence against respiratory bacterial infections relevant to chronic pulmonary disease are less understood. Nontypeable Haemophilus influenzae (NTHi), an opportunistic respiratory pathogen, frequently colonizes the lower airways of patients with chronic lung diseases and is associated with poor clinical outcomes. Human ex vivo lung explants were infected with a clinical strain of NTHi under normoxic and hypoxic conditions for 24 hours. The host response to infection and the effect of hypoxia were investigated using RNA sequencing. Overall design: RNA quality was assessed using an Agilent Bioanalyzer 2100 system before sequencing of enriched poly-(A)-tailed mRNA, which was conducted by Novogene (Cambridge). Libraries were generated using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA), and their quality was evaluated on the Agilent Bioanalyzer 2100 system. Quantification of the libraries was performed using a Qubit 2.0 fluorometer (Life Technologies). Sequencing was executed on the NovaSeq 6000 Illumina® platform, generating 150-base pair (bp) paired-end reads at a sequencing depth of 40 million reads per sample. To ensure high-quality data reads containing adapter sequences, poly-N stretches, and low-quality reads were removed, resulting in clean reads. The Spliced Transcripts Alignment to a Reference (STAR) software (version 2.5) was utilized for mapping the reads to the human genome (GRCh38), with reads quantified using HTSeq (Anders et al., 2015).