Illumina NovaSeq 6000 paired end sequencing; RNA-Seq of WT Flp-In T-REx 293, UPF3A knockout and UPF3A overexpression cell lines in part with additional siRNA-mediated control or UPF3B knockdowns
RNA-Seq of WT Flp-In T-REx 293, UPF3A knockout and UPF3A overexpression cell lines in part with additional siRNA-mediated control or UPF3B knockdowns
A006850100_136530_p
Flp-In 293 T-REx (Thermo Fisher Scientific) WT, UPF3B-KO and UPF3A-UPF3B dKO 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 2 x 10^5 cells using 2.5 μl Lipofectamine RNAiMAX. To induce expression of the stably integrated UPF3A construct, 30 μg/ml cumate was added and the cells were harvested after 72h. Total RNA was extracted using the Direct-zol RNA MiniPrep kit including the recommended DNase I treatment (Zymo Research) 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.