GSM5582711: BN_Gt033; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated from cytapheresis packs by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, NJ, USA). For isolation of specific immune cells, total B, CD4 and CD8 cells were enriched by negative selection using EasySep Negative Human kits specific for each cell type; monocytes were negatively enriched using “EasySep Human Monocyte Enrichment Kit w/o CD16” (StemCell Technologies, Vancouver, Canada) following the manufacturer's instructions. Natural killer cells were negatively enriched by depleting PBMCs with antibodies directed against CD3, CD4, CD14, CD19 and Glycophorin-A in HBSS buffer. Enriched cell populations were further purified by flow cytometry following Human Immunophenotyping Consortium (HIPC) phenotyping panels (Maecker et al., 2012).  Gating strategies and post-sort purity were analyzed by FlowJo software. Granulocytes were positively selected from whole blood using EasySep™ Human Whole Blood CD66b Positive Selection Kit Total RNA was prepared from 2 million cells using miRNeasy mini kit (Qiagen Inc, CA) according to the manufacturer's recommendations. RNA quality and quantity were checked using RNA-6000 nano kits on the Agilent 2100-Bioanalyzer and 500ng of the total RNA was used for ribosomal RNA (rRNA) depletion using GeneRead rRNA Depletion Nano Kit (Qiagen Inc, CA). 50ng of the rRNA depleted RNA was used for cDNA synthesis followed by single primer isothermal amplification (SPIA) using Ovation RNA–Seq System V2 kits according to manufacturer's protocol (Nugen Technologies Inc, CA). 375ng of amplified cDNA sheared to an average size of 150–250 bases, was used to prepare the libraries using Illumina ChIP-Seq kits (Illumina Inc, CA) according to the manufacturer's protocol, followed by sequencing using Illumina Hiseq2500 sequencer with V4 reagents