characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-08-13T00:18:59Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-08-13T00:18:59Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110328959
characteristics.characteristic: External Id
characteristics.text: LabMedMolGe
characteristics.characteristic: INSDC center name
characteristics.text: 2023-08-13T00:18:59Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-08-13T00:18:59Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12426043
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11900:Sample 2
characteristics.characteristic: Submitter Id
characteristics.text: 52
characteristics.unit: year
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: A498
characteristics.characteristic: cell_line
characteristics.text: epithelial cell
characteristics.characteristic: cell_type
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: 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)
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: carcinoma
characteristics.characteristic: disease
characteristics.text: praja2RM
characteristics.characteristic: genotype
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: kidney
characteristics.characteristic: organism part
characteristics.text: praja2 overexpression
characteristics.characteristic: phenotype
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.characteristic: sex
characteristics.text: Sample 2
characteristics.characteristic: title