RNA-seq of engineered HEK-293-EBNA cell lines expressing standard, mixed, or immunoproteasome
RNA-seq of engineered HEK-293-EBNA cell lines expressing standard, mixed, or immunoproteasome
Cells were grown in antibiotic-free medium for 2 days prior to harvesting for RNA extraction. The four HEK-293 clones were grown in Iscove׳s Modified Dulbecco׳s Medium (IMDM, ThermoFisher Scientific Cat#21980-032) containing 10% Fetal Bovine Serum (FBS, ThermoFisher Scientific Cat#F7524). The media was supplemented with 600 µg/ml of hygromycin (Sigma-Aldrich Cat#10843555001) for the culture of HEK-293 β1i-β2i-β5 clone, with 5 µg/ml of puromycin (InvivoGen Cat#Ant-Pr5) and 600 µg/ml of hygromycin for the culture of HEK-293 β1i-β2i-β5i clone and 600 µg/ml of hygromycin and 2.5mg/ml G418 (Sigma-Aldrich Cat#4727878001) for the culture of HEK-293 β1i-β2i-β5t clone. All culture media were supplemented with GlutaMAX (ThermoFisher Scientific Cat#35050-038) and 100 U/ml penicillin and 100 µg/ml streptomycin (ThermoFisher Scientific Cat#15140122).  The production of the HEK293-EBNA cells stably expressing thymoproteasome was performed by transfecting HEK-293-EBNA cells expressing the standard proteasome with plasmids encoding the β1i and β2i subunits, as described (Guillaume, Chapiro et al. 2010). Stable transfectants, selected in 600μg/ml hygromycin, were then cloned by limiting dilution. One of the clones obtained (clone 13), which only expresses the proteasome β1i, β2i and β5 subunits, was further transfected with a FLAG-tagged β5t construct cloned in pcDNA3.1(+), previously generated and used in (Kuckelkorn, Stubler et al. 2019). In this construct, the FLAG sequence was added at the 3'-end of the huPSMB11 coding sequence. The recombinant β5t subunit contained a flag sequence to facilitate the detection and immunoprecipitation, and the β5t-flag catalytic activity has previously been investigated (Kuckelkorn, Stubler et al. 2019). The transfection was carried out by LT-transit (Mirus) according to manufacturer's instructions. Transfected cells were selected with 5 mg/ml G418, cloned by limiting dilution, and expression of the corresponding proteasome subunits was verified by western blot for the selected clone. The HEK-293 clone expressing the immunoproteasome was already generated and characterized in (Guillaume, Chapiro et al. 2010, Abi Habib, De Plaen et al. 2020). Two days prior RNA extraction, antibiotic-free medium was used to grow the cells. RNA was extracted using the Nucleospin RNA kit from Macherey Nagel. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.