RNA-seq of engineered T-REx-293 cell lines expressing standard or immunoproteasome
RNA-seq of engineered T-REx-293 cell lines expressing standard or immunoproteasome
Cells were grown in antibiotic-free medium for 2 days prior to harvesting for RNA extraction. We used human embryonic kidney 293 cells expressing the tetracycline repressor (T-Rex-293; Invitrogen Life Technologies) as recipient cells for stable transfection of inducible expression plasmid pcDNA4/TO, which contained, under the control of a CMV promoter and a tetracycline-resistance operon, a tricistronic construct containing the cDNA of human proteasome subunit β1i, the internal ribosome entry site of hepatitis C virus (kindly provided by A-M. Delisse and T. Cabezon, GlaxoSmithKline, Rixensart, Belgium), the cDNA of proteasome subunit β2i, the internal ribosome entry site of Theiler virus (kindly provided by T. Michiels, Institute of Cellular Pathology, Brussels, Belgium), and the cDNA of proteasome subunit β5i. Transfected cells were selected with bleocin (60 μg/ml) (Calbiochem), and further transfected with plasmid pEF/myc/cyto (Invitrogen Life Technologies) containing β2i and plasmid pEF/Bos/Puro (Invitrogen Life Technologies) containing β5i. They were selected alternatively with 3.5 μg/ml neomycin (Promega) and 1 μg/ml puromycin (Sigma-Aldrich). Transfected clone 6, referred to in this study as 293-IP, contained, upon tetracycline exposure, proteasomes bearing only the immunosubunits.  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.