title: Illumina HiSeq 2500 sequencing; RNA-seq of human peripheral blood mononuclear cells (PBMCs) of  the patients with liver transplantation before and after surgery
design: RNA-seq of human peripheral blood mononuclear cells (PBMCs) of  the patients with liver transplantation before and after surgery
library_construction_protocol: Peripheral blood collected in the EDTA anticoagulant tubes (~4 ml) was transferred into Human peripheral blood lymphocyte separation tubes (Dakewe, Shenzhen, China). Then the tubes were centrifuged at 400 g for 20 min at room temperature. After centrifugation, the blood was divided into 4 layers (from top to bottom: plasma layer, mononuclear cell layer, separation liquid layer, and red blood cell layer). Slowly soak out the mononuclear cells that were washed three times with PBS. The isolated PBMCs were stored at -80 °C or frozen in liquid nitrogen. Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer's protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel.  electrophoresis Eukaryotic mRNA was enriched by Oligo(dT) beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-ZeroTM Magnetic Kit (Epicentre, Madison, WI, USA). Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and  buffer.