Hush 29 mer shrna expression vectors

U2OS (ATCC), Flp-In T-Rex HEK293 (Thermo Fisher), Flp-In T-Rex U2OS (a generous gift from Dr Blerta Xhemalce, University of Texas at Austin) and Flp-In HeLa S3 (graciously provided by Dr Till Bartke, Imperial College London) cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% (v/v) Fetal Bovine Serum (Sigma, St Louis, MO, USA) and antibiotics (10 units/ml penicillin and 10 μg/ml streptomycin, ThermoFisher, Waltham, MA, USA) at 37°C and 5% CO₂. For transient siRNA knockdown, reverse transfection of 10 nM targeting or non-targeting control siRNA was performed using jetPRIME (Polyplus-transfection, Illkirch-Graffenstaden, France) following the manufacturer’s instruction, followed by incubation for 48–72 h before downstream analysis. Stable U2OS shRNA knockdown cells were generated by transfection with HuSH 29-mer shRNA expression vectors (Origene, Rockville, MD, USA) using GenJet U2OS Transfection Reagent (SignaGen, Rockville, MD, USA) followed by selection with puromycin (InvivoGen, San Diego, CA, USA) 48 h post-transfection until colonies formed. Colonies were then isolated, expanded and screened for knockdown by western blot and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR).