NextSeq 500 paired end sequencing; Downregulation of RING ligase praja2 boosts receptor tyrosine kinases in kidney cancer
Downregulation of RING ligase praja2 boosts receptor tyrosine kinases in kidney cancer
Kidney cancer line (A498, ATCC) were cultured in Dulbecco modified Eagle's medium, containing 10% fetal bovin serum in an atmosphere of 5% CO2. Human Kidney Carcinoma Cell Line (A498, and A498 over-expressing praja2 WT or praja2RM under a tetracycline-inducible promoter), were maintained in DMEM medium (Gibco) supplemented with 10% Fetal Bovine Serum, NCS (Sigma-Aldrich), 1% L-glutamine, 1% penicillin/ Sigma-Aldrich). Inducible lentiviral vectors, were generated using the In-Fusion® HD Cloning Kit technology. praja2 CDSs were amplified by PCR using Origene plasmids vectors as template, and cloned in PCW57.1 vector (Addgene #41393) digested Bam HI. A498 cells were transfected by calcium-phosphate, using a DNA mix containing plasmid for over-expression (12 µg) harboring or not praja2 CDSs and psPAX2 (Addgene #12260) (18 µg) and pMD2.G (Addgene #12259) (13 µg) as packaging vectors. Forty-eight hours post-transfection, cell supernatants were collected, filtered through a 0.45 µM filter, and 10 µg/mL of polybrene (Merk) was added. A498 cell media was then replaced with infective media, and cells were incubated for 6 h. Later, the infective cell medium was replaced with normal medium and, after an additional 24 h, 1 µg/mL of puromycin was added to start selection. The day after the transfection, cells were induced (using 1 µg/ml of doxycycline for 72h) or not, then cells were collected and lysed for Western blotting analysis. RNA purity and integrity were assessed with a Nanodrop 2000c spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and a 4200 TapeStation instrument (Agilent Technologies, Santa Clara, CA, USA), respectively. For RNA purity, an A260/280 ratio of 2.0 and an A260/230 ratio of 2.0-2.2 were considered acceptable; for RNA integrity, an RNA Integrity Number (RIN) of 9.0-10.0 has been obtained for all samples, indicating the absence of degradation and high integrity of RNA samples. For a precise estimation of the RNA concentration, a Qubit 2.0 fluorometer assay (Thermo Fisher Scientific, Waltham, MA, USA) has been employed. For RNA sequencing, 1mg of high-quality total RNA was used for library preparation with a TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA, USA)