Turbofect transfecting reagent

HEK293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen) at 37°C, 5% CO₂. For transient transfection, TurboFect Transfecting Reagent (Thermo Scientific) was used according to the manufacturer's recommendations. GFP-fused ARS2 FL, fragments F1-F6 and mutants DUF, ZnF, M and ΔC were stably expressed in a HEK293 Flp-In T-REx tetracyclin-inducible cell line. Fusion proteins were introduced using a modified pcDNA5/FRT/TO vector that contains an N-terminal GFP-tag followed by the protein of interest. HEK293 0-LpA and Histone cell lines were described in (31 (link)). All HEK293 Flp-In T-REx tetracyclin-inducible cell lines were maintained in DMEM containing 10% tetracycline-free FBS (Invitrogen), 1% penicillin/streptomycin, 5 μg/ml Blasticidin (Invitrogen) and 100 μg/ml Hygromycin B (Invitrogen).

For RNAi assays, cells were seeded in Collagen (Sigma) pre-coated dishes and grown for 24 h before the first transfection. siRNA transfections were performed using siLentFect Lipid Reagent (Bio-Rad) according to the manufacturer's instructions and siRNAs at a final concentration of 20 nM. Forty eight hours after the first transfection, media was changed and the transfection was repeated. At least 5 h after each transfection, media was replaced with fresh media. Cells were harvested 24 h after the second transfection. siRNA sequences are listed in Supplementary Table S3. Plasmids transfections were performed using Turbofect Transfecting Reagent (Thermo Scientific) according to the manufacturer's recommendations, 48 hours before the cell harvest. Prior to cell harvest, plasmid transfection efficiencies were monitored by fluorescence microscopy and found to be constant between 70 and 80%.