Dmrta2 specific sirna

LN18 spheres cultured for 10 days or WG14 spheres cultured for 21 days were collected by centrifugation, washed with PBS and dissociated to single-cell suspension using TrypLe Express (Thermo Fisher Scientific). 1 × 10⁵ cells were transfected with 50 nM of control or DMRTA2-specific siRNA (Dharmacon) in 20 μl of SF buffer (SF Cell Line 4D-Nucleofector ^TM X Kit) using 4D-Nucleofector system (Lonza). After transfection, cells were immediately resuspended in a fresh medium for spheres without antibiotics, plated on 60 mm plates for cell suspension and incubated for 24 h.

LN18 glioma cells were seeded on 96-well microplates (at the density of 4 ×10³/well) for MTT metabolism and BrdU incorporation assays and 6-well plates (at the density of 2.25 × 10⁵/well) for total RNA and protein isolation and left overnight to attach. The next day, the culture medium was removed and cells were transfected with 25 nM control or DMRTA2-specific siRNA (Dharmacon) using Viromer® BLUE transfection reagent (Lipocalyx GmbH, Halle, Germany). The culture medium was replaced after 4 h. The cells were assessed for cell viability and proliferation or collected for total RNA and protein isolation for Western Blot or harvested and seeded at a low density for the sphere formation assay. The efficiency of gene knock-down was determined by qPCR and Western blot analysis.