CG4928_KD 3
mRNA was separated from total RNA using Dynabeads mRNA Purification kKit (Thermo Scientific) according to the manufacturer's protocol. Poly-A selected RNA was treated with RNaseIII according to the Ion Total RNA-Seq protocol (Thermo Fisher) and purified with Magnetic Bead Cleanup Module (Thermo Fisher). The size and quantity of RNA fragments were assessed on the Agilent 2100 Bioanalyzer system (RNA 6000 Pico kit, Agilent Technologies) before proceeding to library preparation, using the Ion Total RNA-Seq kit for the AB Library Builder System (Thermo Fisher). The libraries were amplified according to the protocol and purified with Magnetic Bead Cleanup Module (Life Technologies). Samples were then quantified using the Agilent 2100 Bioanalyzer system (High Sensitivity DNA kit, Agilent Technologies) and pooled, followed by emulsion PCR on the Ion OneTouch 2 system using the Ion PI Hi-Q OT2 200 chemistry (Thermo Fisher). Enriching was conducted using the Ion OneTouch ES (Thermo Fisher). Samples were loaded on an Ion PI v3 Chip (3 samples per chip) and sequenced on the Ion Proton System using Ion PI Hi-Q Sequencing chemistry (200 bp read length, Thermo Fisher).