Genome wide transcriptomic experiments: gene counts for whole blood RNA-Seq from children with septic shock at PICU admission (TP1) and 48 hours later (TP2)

Gene Expression Analysis for Modelling Immune Trajectory and Endotype Transition During Pediatric Septic Shock We aimed to characterise the immune trajectory of 19 children with septic shock from first interaction with pediatric critical care teams (TP1) to 48 hours afterwards (TP2). We analysed data collected as part of the Biomarkers of Acute Serious Illness in Children (BASIC) study that recruited participants between 2014–201623. BASIC was a prospective cohort study that enrolled critically ill children admitted to four PICUs in London and Eastern England, UK, during emergency transport by the Children’s Acute Transport Service (CATS), the regional paediatric critical care retrieval service. We therefore selected 19 children with septic shock (defined operationally as children with suspected or confirmed infection who were invasively ventilated and receiving vasoactive infusions via a central venous catheter) from whom TP1 and TP2 RNA-stabilised samples were available. Whole blood was sampled into PAXgene (Qiagen, UK) tubes and stored appropriately at -80ºC. RNA was extracted according to manufacturer’s instructions using the QIAsymphony PAXgene blood miRNA extraction kit (Qiagen, UK). Extracted samples of RNA were DNA depleted (RNA Clean and Concentrator-5 with DNase, Zymo Research, CA, USA) and globin depleted (GLOBINclear Human Kit, ThermoFisherScientific) and sequenced on an Illumina NextSeq2000 at a core genomics facility (Imperial Genomics Facility, Imperial NIHR Biomedical Research Centre) to generate 50 bp paired-end reads. Raw fastq files were aligned to the human genome (GRCh38) using Salmon 1.10.2 (https://salmon.readthedocs.io/en/latest/salmon.html)45 and gene counts quantified prior to differential gene expression analyses. Downstream analyses included: differential gene expression analysis; weighted gene co-expression network analysis; endotyping (using previously published BASIC endotypes); and RNA velocity and transcriptomic trajectory methods. The code for downstream analyses can be found via the published manuscript, or at: https://github.com/michaeljamescarter/basic_trajectory or https://github.com/DrClaireDunican/VeloCD.