Qiacube automated system

Viral RNA was extracted from clarified stool specimens (10–20% w/v) using an automatic RNA extraction procedure according to the manufacturer’s instructions (QIAcube^® Automated System and QIAamp^® Viral RNA Mini kit; Qiagen, CA, USA). RVA detection was performed using RT-qPCR on an Applied Biosystems® 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as previously described^{51 (link)}. The RVA RT-qPCR result was considered positive if the cycle threshold (Ct) value was ≤ 40.

Total gDNA was extracted from an overnight culture (2 ml) on a QIAcube automated system (Qiagen). Following extraction, gDNA was quantified by fluorometric methods using a Qubit (ThermoFisher Scientific), with quality ratios of gDNA (A260/280 and 260/230) determined via Nanodrop (ThermoFisher Scientific). Genomic DNA libraries are prepared for whole-genome sequencing using the NexteraXT kit (Illumina), as described by the manufacturer. Paired end sequencing was performed using the Illumina MiSeq platform (MiSeq Reagent V3 Kit; 2 × 300 cycles). For each E. coli isolate, at least 80× coverage was generated. Raw sequence reads were trimmed using Trim Galore and the genomes were de novo-assembled into contigs using SPAdes (3.9.0) with pre-defined kmers set. Raw reads were also assembled with Geneious (10.0.9; Biomatters Ltd.) de novo assembler, set at medium sensitivity for analysis of paired Illumina reads. Geneious was used to map both sets of contigs to reference genes identified by closest BLAST homology and was also used to annotate genes from closest homologues in NCBI Genome database. Resistance genes were identified using Resfinder within CGE^{59 (link)}, and wgMLST profiles were generated using the CGE platform coupled with the PubMLST.org database^{60 (link)}. Plasmids were identified within the genome assembly and typed using Plasmidfinder^{61 (link)}.

Viral RNA was purified from stool samples stored at –20°C. A 140 μL suspension (10% w/v) of each stool sample was prepared with Tris-calcium buffer (pH = 7.2) and subjected to an automatic RNA extraction procedure using a QIAamp^® Viral RNA Mini kit (QIAGEN, CA, USA) and a QIAcube^® automated system (QIAGEN), according to the manufacturer’s instructions. Part of the isolated nucleic acid was transcribed to cDNA using a High Capacity cDNA Reverse Transcription Kit (Life Technologies™, NY, USA), and an aliquot was immediately stored at −80°C. In each extraction procedure, RNAse/DNAse-free water was used as negative control.

Whole blood was collected in PAXgene tubes (Becton‐Dickinson) within 24 h after ICU admission and total RNA was isolated using the PAXgene blood mRNA kit (Qiagen, Venlo, the Netherlands) in combination with QIAcube automated system (Qiagen), as previously described.¹³, ¹⁸, ¹⁹ The RNA isolation excluded material from erythrocytes. Microarray data (Affymetrix Human Genome U219 96‐array plates) are accessible to the public via the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) accession GSE65682. Briefly, raw scans were pre‐processed by means of the robust multi‐average (RMA) method, normalized (quantile), summarized by median polish and log2 transformed using the affy method.²⁰ Nonexperimental chip effects were assessed and corrected by means of the combat method in the surrogate variable analysis R package.²¹ Comparisons between groups were done using multi‐variate linear models, including age and gender as covariates, implemented in the limma method.²² Benjamini–Hochberg (BH)‐adjusted p‐values <0.05 defined genome‐wide significance. To assess the association with canonical signalling pathways, we used Ingenuity Pathway Analysis software (Qiagen Bioinformatics). Fisher exact test BH‐adjusted p‐values <0.05 demarcated significance. Human species and Ingenuity gene knowledgebase were specified. All other parameters were default.

RNA was extracted from cell pellets with the RNeasy mini kit (Qiagen, Hilden, Germany) using the Qiacube automated system (Qiagen); 1 µg RNA from each sample was taken to reverse transcription reaction. Next, cDNA was generated with High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA, USA). Quantitative real-time polymerase chain reaction (qPCR) was performed using real time PCR (Biometra Analytik, Jena, Germany) and SYBR Green (Fast SYBR™ Green Master Mix, Applied Biosystems™, Foster city, CA, USA). Syntezza Bioscience (Jerusalem, Israel) supplied all the primers (SDF1, VEGF-A, CCL2, PDGFβ, VEGFR-2, and CXCR4). As an internal control, levels of HPRT were quantified in parallel with target genes (Table S2, primers’ sequence). Results were analyzed using Profiler PCR Array data analysis tool (qPCRsoft3.2, Analytik Jena, Germany) and normalized to generate fold change for each gene using the ΔΔCt method [22 (link)].

Genomic DNA was isolated from whole blood using the QIAcube automated system (Qiagen). SNPs were selected based on their putative role as cytokine QTLs in the HFGP study and on their ability to tag surrounding variants with a pairwise correlation coefficient r² of at least 0.80 and a minor allele frequency of ≥5% using publicly available sequencing data from Pilot 1 of the 1000 Genomes Project for the CEU population. Genotyping of rs674430 and rs646564 SNPs in PFKFB3 was performed using KASPar assays (LGC Genomics) in an Applied Biosystems 7500 fast real-time PCR system (Thermo Fisher Scientific), according to the manufacturer’s instructions. The DNA samples of individuals from the 500FG cohort were genotyped using the commercially available chip Illumina HumanOmniExpressExome-8 v1.0. Quality control and imputation were performed as described previously (43 (link)).