GSM6934605: HC_14; Homo sapiens; RNA-Seq
Total RNA was isolated from peripheral blood (PAXgene blood RNA kit, Qiagen), concentration was measured by Qubit fluorimetr and RNA integrity number was checked using Agilent Bioanalyzer 2100. Isolated RNA (800ng) was rRNA and globin depleted using NEBNext® Globin & rRNA Depletion Kit (New England, BioLabs, Inc). Transcriptome libraries for differential gene expression were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads according to the manufacturer's protocol (New England, BioLabs, Inc). In brief, depleted RNA was randomly sheared by heat digestion in the presence of a divalent metal cation (Mg2+). Sheared RNA was reversibly transcribed making 1st strand of cDNA using random hexamers as primers and reverse transcriptase. The second strand was created using dUTPs and after purification with Sample Purification Beads, NEBNext adapters were ligated. After removal of 2nd strand by uracil-DNA-dependent glycosylase, final amplification of adaptor ligated DNA using NEBNext® Multiplex Oligos for Illumina® was done. Library quality was assessed on a Bioanalyzer 2100 using Agilent DNA 1000 assay.