Oligofectamine 2000

HeLa cells were grown in Eagle's MEM plus 10% fetal calf serum, 2 mM l-glutamine, 100 U·mL⁻¹ penicillin, 100 µg·mL⁻¹ streptomycin and 1% nonessential amino acids (Sigma, St Louis, MI, USA), and incubated in humidified air containing 5% CO₂ at 37°C. At 80% confluence, cells were transfected with different DNA constructs using Oligofectamine 2000 (ThermoFisher, Waltham, MA, USA) as a transfection reagent and incubated for 24 h before harvesting.

HEK293 and HeLa cells (from Life Technologies) and MHT cells (50 (link)) were grown in DMEM medium supplemented with 10% FBS at 37°C in an incubator with 5% or 8% CO₂. One day before transfection, cells were seeded at ∼60% confluency and incubated overnight. Transfection of ASOs was performed using Oligofectamine 2000 or RNAiMax (Life Technologies). For the RNASEH1 cleavage activity assay, cells transfected with ASO761919 were reseeded at ∼60% confluency, allowed to grow for 14 h, and then transfected again with an ASO targeting Malat1 (ASO395254). RNA was harvested 4 h later. Transfection with siRNAs (3–5 nM final concentration) was performed using RNAiMax (Life Technologies). At 4 h or 24 h after siRNA transfection, cells were washed and transfected with ASOs. For the attenuation study using complementary oligonucleotide (Figure 1), HEK293 cells were co-transfected with ASO761919 and ASO927728 or with ASO761919 and ASO759704.