Hiscan sq scanner

Genomic DNA (1 μg) was bisulfite-converted using the EZ-96 DNA Methylation Kit (Zymo Research, Orange, CA, USA) according to the manufacturer's procedure and recommended alternative incubation conditions when using the Illumina Methylation Assay.
Genome-wide DNA methylation was assessed using the Illumina HumanMethylation450 Beadchip (Illumina Netherlands, Eindhoven, Netherlands) following the manufacturer's protocol with no modifications. The arrays were scanned with the Illumina HiScan SQ scanner. These processes were carried out in Progenika Biopharma in Bizkaia, Spain.

The Illumina Infinium HumanMethylation450 Beadchip Kit (Illumina Inc., San Diego, CA, USA) was utilized for generation of methylation data for all samples. The analysis was done according to standard protocol provided by Illumina. Bisulfite conversion was done using a Zymo Research EZ DNA methylation kit (Zymo Research, Irvine, CA, USA). Beadchips were scanned with an Illumina HiScanSQ scanner using standard settings. Initial quality control, background subtraction and raw data normalization were done using the standard algorithms provided in Illumina Genome studio Methylation module v1.0. Methylation levels are quantified using β-values, as recommended by Illumina. Briefly, the β-value for each interrogated CpG site represents the fraction of methylated versus non-methylated probes, and consequently, the β-value ranges from 0 (unmethylated) to 1 (fully methylated). All analysis and statistical testing was performed on β-values.

Retrospectively, the total DNA of 16 patients (critically ill non-septic patients (n = 4), septic patients (n = 7) and SS patients (n = 5) was isolated from the total blood cell pellet stored at the INCLIVA biobank with the All-In-One DNA/RNA Miniprep Kit (BS88203, Bio Basic Canada Inc (Markham, ON, Canada)) following the manufacturer’s instructions. Purified DNA was quantified with the fluorometric method (Quant-iT PicoGreen dsDNA Assay, Life Technologies, Carlsbad, CA, USA). Genome-wide methylation was assessed as previously described by Pozo-Lorente et al. [21 (link)] using Infinium Human DNA Methylation EPIC 850 K arrays (Illumina Inc., San Diego, CA, USA) scanned on an Illumina HiScan SQ scanner (Illumina Inc., San Diego, CA, USA). The minfi R-package [22 (link)] was used to process and normalize the arrays [22 (link),23 (link)].
The proportions of the different immune cells were obtained using the estimate Cell Counts 2() function of the FlowSorted.Blood.Epic package [24 (link)] using a process of deconvolution (Identifying Optimal Libraries—IDOL) based on discriminating differentially methylated regions (DMRs) specific to each cell type [25 ]. The neutrophil-to-lymphocyte ratio (NLR) was calculated taking the ratio between the estimated cell proportion of neutrophils and the estimated cell proportions of CD4T, CD8T, NK and B cells.