GSM4477714: 33_0M; Homo sapiens; RNA-Seq
Whole blood was collected with PAXgene blood RNA tubes (Qiagen). Silica-membrane-based RNA isolation and purification in a spin-column format was performed using PAXgene Blood RNA kit (Qiagen). Extracted RNA was globin-cleared using the Ambion GLOBINclear kit (Thermo Fisher, Waltham, MA). 500 ng total RNA was rRNA-depleted using NEBNext rRNA Depletion Kit (New England Biolabs) and converted to an Illumina sequencing library using NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). Libraries were validated with Bioanalyzer (Agilent Technologies) to determine size distribution and concentration. Sequencing was performed by Tsukuba i-Laboratory LLP (Tsukuba, Japan) using NextSeq500 (Illumina) with paired-end 36-base read option. Sequencing reads were mapped on the hg19 human reference genome and quantified using CLC Genomics Workbench (CLC-GW) version 10.1.1 (Qiagen). Differentially expressed genes were identified by filtering according to p-value obtained by Empirical Analysis of DGE tool in CLC-GW. RNA libraries were prepared for sequencing using standard Illumina protocols