GSM6774717: HD003_Healthy; Homo sapiens; RNA-Seq
Venous blood was collected from subjects into ethylenediaminetetraacetic acid tubes.  Peripheral blood monocular cells (PBMCs) were isolated using SepMate tubes (STEMCELL Technologies) according to the manufacturer's instructions.  Briefly, blood was diluted with phosphate buffered saline (PBS; Gibco) after plasma collection.  The diluted blood was then layered on top of Ficoll-Paque PLUS (Merck).  After centrifugation (1200 g, 10 min, brake on), the supernatant was poured into a 50 mL polypropylene tube.  Collected PBMCs were washed twice in PBS.  PBMCs were counted using a hemacytometer with trypan blue (Lonza) to determine cell viability.  Ribonucleic acid (RNA) was prepared using the PureLink RNA Mini kit (Thermo Fisher Scientific). The total RNA was quantified using Qubit Fluorometer (ThermoFisher Scientific).  The purity was checked using NanoPhotometer spectrophotometer (Implen).  The integrity and quality of the RNA was assessed using Agilent Bioanalyzer 2100 system (Agilent Technologies) and agarose gel electrophoresis (1% agarose gel, 180 V, 16 min).  A total of 2 µg RNA from each sample was subjected to ribosomal RNA (rRNA) removal using Epicentre RiboZeroTM rRNA Removal Kit (Epicentre). The rRNA was cleaned up using ethanol precipitation method.  The Next Generation Sequencing (NGS) library was prepared using NEBNext UltraTM Directional RNA Library Prep Kit for Illumina (NEB).  In brief, the RNA were fragmented using divalent cations at 94°C in the presence of NEBNext First Strand Synthesis Reaction Buffer and Random Primers.  Subsequently, the first strand complementary deoxyribonucleic acid (cDNA) was synthesized using random hexamer and M-MulV reverse transcriptase and the second strand cDNA was synthesized using DNA Polymerase and RNAse H.  The dTTP was replaced by dUTP during the second strand cDNA synthesis. The double stranded cDNA fragment was converted to blunt end via end repair reaction and the NEBNext adapter (hairpin loop structure adapter) was ligated to the adenylated cDNA fragment using ligase.  Next, the second-strand cDNA was degraded with the aids of USER enzyme.  Size selection and sample purification was performed using AMPure XP Beads (Beckman Coulter).  Subsequently, Illumina sequencing adapter and indexes were added onto the cDNA fragment via PCR amplification (hereafter known as sequencing library).  The sequencing library was purified using Ampure XP Beads (Beckman Coulter).  The size distribution of the sequencing library was assessed using Agilent Bioanalyzer 2100 system (Agilent Technologies). The qualified sequencing library was loaded onto NovaSeq 6000 (Illumina) for sequencing using paired-end 150 sequencing strategy.  Raw data generated from the sequencer were demultiplexed and converted to FASTQ file using Illumina bcl2fastq.