title: GSM2792922: ZFAS1_ORF_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using RNeasy kit (Qiagen, GE) as recommended by the manufacturer. Polyadenylated RNA was isolated from total RNA using standard protocols (Dynabeads mRNA Direct Micro Kit, Ambion/Life Technologies, USA) starting with 5 μg total RNA. Library preparation for NGS using Ion Torrent technology (Life Technologies, USA) was carried out according to the manufacturers recommendations (Ion Total RNA-Seq Kit v2, Ion torrent/Life Technologies, USA), and sequenced using an Ion Proton system (Life Technologies, USA). Quality control, quantification of RNA and libraries was carried out using using Agilent RNA 6000 Nano Kit or Agilent High Sensitivity DNA kit and Agilent Bioanalyzer (Agilent Technologies, USA). In brief, approximately 50 ng polyA RNA was fragmented down to 100-300 base fragments using RNaseIII for 1-3 min followed by adapter ligation, amplification for 9-14 cycles and barcoding using Ion Express RNA-Seq Barcode kit (Ion Torrent/Life Technologies, USA). The final library fragment size and concentration was determined by Agilent Bioanalyzer analysis followed by template preparation using Ion PI Template OT2 200 Kit v3 (Life Technologies, USA) and Ion One Touch System followed by NGS on an Ion Proton system using Ion PI™ Chip Kit v2. In general two transcriptome libraries were barcoded and analyzed on one Ion PI v2 chip.