Illumina NovaSeq 6000 paired end sequencing; RNA-Seq of control and EJC factor (EIF4A3, RBM8A, MAGOH) knockdowns in part with EIF4A3 rescue in HeLa Tet-Off cells
RNA-Seq of control and EJC factor (EIF4A3, RBM8A, MAGOH) knockdowns in part with EIF4A3 rescue in HeLa Tet-Off cells
HeLa Tet-Off (Clontech) 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 EIF4A3, RBM8A or MAGOH+MAGOHB (or Luc in the negative control) per 2-3 x 10^5 cells using 2.5 μl Lipofectamine RNAiMAX. Total RNA was extracted using peqGOLD TriFast (VWR Peqlab), followed by using the DIRECTzol Miniprep Kit (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.