title: GSM5596128: pNK4; Homo sapiens; RNA-Seq
library_construction_protocol: NK cells were isolated, flash frozen on dry ice, and RNA was harvested using Trizol reagent. 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. RNA libraries were prepared for sequencing using standard Illumina protocols:  After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-Zero TM  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. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, A base added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).