*4.11. Analysis of Cell Cycle and Cell Death Using Fluorescence-Activated Cell Scanning (FACS) Flow Cytometry*

For FACS based analysis of the cell cycle changes upon *LINC01133* knockdown, we used standard propidium iodide (PI) DNA staining protocol. In brief, transfected cells were harvested 72 h after siRNA knockdown and 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells were fixed in 70% precooled ethanol for 2 h on ice. After washing with PBS (Thermo Fisher Scientific, Waltham, MA, USA) the cells were re-suspended in 0.5 mL PI/RNAse containing staining buffer (550825), BD Pharminogen™, Heidelberg, Germany) supplemented with 10 µL PI staining solution (51-6621-1E, BD Pharmingen™, Heidelberg, Germany). After incubation for 15 minutes at room temperature, the number of PI positive cells was measured by flow cytometry. The effect of *LINC01133* on cellular apoptotic rate was evaluated using staining with AnnexinV (FITC-conjugated antibody (640906), BioLegend, San Diego, CA, USA; Annexin V Binding Buffer, (422201), BioLegend, San Diego, CA, USA) in conjunction with the vital dye 7-amino-actomycin D (00-6993-50) eBioscience, San Diego, CA, USA) followed by flow cytometry measurement. A total of 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells 72 h after transfection was used in the analysis, with a total of 10,000 events recorded for each sample, and unstained cells being used as assay control.
