GSM6690539: GFHD112, DMSO-Pg20, 4h; Homo sapiens; RNA-Seq
Total RNA was extracted using an RNeasy Mini Kit (Qiagen) mRNA was enriched using oligo(dT) beads from NEBNext® Poly(A) mRNA Magnetic Isolation Module (cat# E7490L; NEB). Next, sequencing libraries were generated using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina® (cat# E7770L; NEB) according to the manufacturer's instructions. Briefly, fragmentation was performed using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using M-MuLV Reverse Transcriptase (RNaseH-) and random hexamer primer. Second strand cDNA was synthesized using DNA Polymerase I and RNase H  and the reaction buffer containing dUTP instead of dTTP. The remaining overhangs were converted into blunt ends by exonuclease/polymerase activities. Following adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified with AMPure XP beads (cat# A63987; Beckman Coulter) to select cDNA fragments of 250-300 bp in length. Subsequently, 3 μl USER Enzyme (NEB) was incubated with size-selected, adaptor-ligated cDNA at 37° C for 15 min followed by 5 min incubation at 95°C. Next, PCR was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified using AMPure XP beads and library quality was assessed using the Agilent High Sensitivity DNA Kit (cat# 5067-4626) on the Agilent Bioanalyzer 2100 system (Agilent Technologies).