RNA-Seq of Homo sapiens: adult PBMC; Acute Non-survivor
RNA from PBMCs was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), quantified using Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific), and integrity checked in a 2200 TapeStation system using RNA ScreenTape (Agilent Technologies, Santa Clara, CA). The cDNA libraries were constructed using the QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina FWD (Lexogen GmbH, Austria), following the manufacturer's protocol. The concentration and the median size of the libraries were assessed by 2200 TapeStation with a DNA1000 ScreenTape (Agilent). The final pool of libraries was quantified by qPCR using the Kapa Sybr Green qPCR Kit (Roche Diagnostics, Mannheim, Germany) and subjected to a single-end sequencing (75 bp) in a NextSeq 500 Sequencing with NextSeq 500/550 High Output Kit v2.5 (Illumina, San Diego, CA). Raw single-end reads were preprocessed for quality control. Sequencing quality was assessed before and after adapter trimming using the program FastQC (Andrews 2010). The Trimmomatic software version 0.39 (Bolger, Lohse, and Usadel 2014) was used to remove adapters sequence trimming the 5, and 3 ends with a mean quality score below 25 (Phred+33) and discard reads shorter than 36 bp after trimming. We used the parameters LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36. After preprocessing, the high-quality reads were mapped into the reference genome Homo sapiens (version GRCh38) with the bowtie2 program (version 2.2.5) (Langmead and Salzberg 2012). To quantify the gene abundance of the mapped reads in each sample, we used the featureCounts tool from the R/Bioconductor package Rsubread (Liao, Smyth, and Shi 2019) with the following parameters: GTF.featureType = gene, GTF.attrType = gene_id, isPairedEnd = FALSE, minOverlap = value 1, allowMultiOverlap = FALSE, countMultiMappingReads = FALSE. Normalization of the gene counts was performed with counts per million normalization (CPM) and quantile normalization, which accounts for differences in library size.