Illumina NovaSeq 6000 paired end sequencing; RNA-Seq of Flp-In T-REx 293, two clones of CRISPR-Cas9 mediated knockout of CASC3 and further CASC3 depletion via small interfering RNAs
RNA-Seq of Flp-In T-REx 293, two clones of CRISPR-Cas9 mediated knockout of CASC3 and further CASC3 depletion via small interfering RNAs
Flp-In 293 T-REx (Thermo Fisher Scientific) WT and CASC3-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 CASC3 (or Luc in the negative control) per 2 x 10^5 cells using 2.5 μl Lipofectamine RNAiMAX.  Total RNA was extracted with NucleoSpin RNA Plus (Macherey-Nagel) 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.