Leukolock

Total RNA was extracted using LeukoLOCK (Thermo Fisher), which captures total RNA from neutrophils, eosinophils, basophils, monocytes, and lymphocytes, and preserved at −80°C. All RNA samples had a RNA Integrity Number (RIN) of at least 6 measured on Agilent Bioanalyzer. Library preparation was performed in batches of up to 96 samples (with multiple samples from the same participant always processed within the same batch) on 1–4 μg total RNA, per standard Illumina TruSeq Stranded mRNA library preparation protocol, and sequenced on Illumina NextSeq500 to a depth of 21 million (M) to 76M reads, mean 41M reads (150 bp paired-end). HISAT2 (Kim et al., 2015 (link)) was used to align demultiplexed reads to the human genome version ‘GRCh37_snp_tran’, which considers splicing and common genetic variants. Aligned and cleaned (deduplicated) reads were counted using HTSeq and GRCh37.75 transcriptome assembly across 63,677 genes. Post-sequencing quality control included removal of samples with excess PCR duplicate rate (>60%) and genotype QC check against respective DNA sample. For all gene expression analyses, genes on sex chromosomes and genes with expression below 6 reads or 0.1 counts per million in at least 20% of samples were dropped. The final RNA-seq dataset consists of 251 unique samples and 18,904 genes.

We obtained samples for gene expression profiling by rapidly isolating the total blood leucocyte population from whole blood samples (about 10 mL) taken following admission to ICU by use of the LeukoLOCK (Thermo Fisher Scientific, Waltham, MA, USA) depletion filter technology (appendix 1 p 2). We purified total RNA and used Illumina Human-HT-12 version 4 Expression BeadChips with 47 231 probes (Illumina, San Diego, CA, USA) to do genome-wide transcription profiling for the first available sample taken following ICU admission for each of 270 patients in the discovery cohort and 114 patients in the validation cohort (appendix 1 p 2). We purified genomic DNA from whole blood samples (appendix 1 p 3) and genotyped individuals in the discovery cohort with Illumina HumanOmniExpress BeadChips (730 525 SNPs; Illumina, San Diego, CA, USA).