title: Illumina HiSeq 4000 paired end sequencing; RNA-Seq of WT Flp-In T-REx 293 and UPF3B knockout cell lines with additional siRNA-mediated control or UPF3A knockdowns
design: RNA-Seq of WT Flp-In T-REx 293 and UPF3B knockout cell lines with additional siRNA-mediated control or UPF3A knockdowns
library_construction_protocol: Flp-In 293 T-REx (Thermo Fisher Scientific) WT and UPF3B-KO cells were maintained at 37° C, 5% CO2 and 90% humidity in Dublecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 9% fetal bovine serum (FBS) and Penicillin-Streptomycin (both Thermo Fisher Scientific). For siRNA transfection, cells were reverse transfected with 60 pmol siRNA targeting UPF3A (or Luc in the negative control) per 3 x 10^5 cells using 2.5 μl Lipofectamine RNAiMAX. Total RNA was extracted using peqGOLD TriFast (VWR Peqlab) according to the manufacturer's instructions. Ribosomal depletion and strand specific library preparation was carried out with TruSeq Stranded Total RNA (with Ribo-Zero Gold) according to the manufacturer's instructions.