PAXGene_2
For whole blood RNA-Seq, blood of patients was collected in PAXgene Blood RNA tubes (BD, Heidelberg, Germany), incubated at room temperature for 2 h to achieve complete lysis of blood cells, and frozen at -80 C until further processing. Before nucleic acid isolation, tubes were thawed at room temperature for 2 h. Nucleic acid isolation was performed using the QIAcube (Qiagen, Hilden, Germany) and the PAXgene blood miRNA kit according to the manufacturers protocol to extract gene-encoding mRNAs. Nucleic acids were eluted in 2x40 L Buffer BR5. The quantity and quality of the isolated RNA was determined with a Qubit Fluorometer (Thermo Fisher, Waltham, Massachusetts, USA) and a Fragment Analyzer (Agilent, Santa Clara, California, USA), respectively. Library preparation and sequencing were performed using 250 ng RNA with the ScriptSeq kit v2 (Illumina, San Diego, CA, USA). Sequencing of the libraries was performed with HiSeq2500 (Illumina, San Diego, CA, USA), with 100 bp single end reagent kits.