biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicstranscription profiling by arrayHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-6748Human renal cell carcinoma cell line 786-O was stably transduced with shKDM5C or empty vector. Then KDM5C was rescued in shKDM5C and over expressed in cells with empty vector. The cells were used to produce tumors in nude mice and were treated with Sunitinib (SU-11248, Pfizer, Inc., New Yourk) at dose of 50mg/kg/day SID.biostudies-arrayexpressScaning - Hybridization is performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed once in High Temp Wash buffer (Illumina Inc.) at 55°C and then twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes (in between washed with ethanol at room temperature). After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays are dried and scanned.Labeling - Biotin-labeled cRNA samples for hybridization on Illumina Human/Mouse/Rat Sentrix-6/8/12 BeadChip arrays (Illumina, Inc.) were prepared according to Illumina's recommended sample labeling procedure based on the modified Eberwine protocol (Eberwine et al., 1992). In brief, 250 – 500 ng total RNA was used for complementary DNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA according to the MessageAmp II aRNA Amplification kit (Ambion, Inc., Austin, TX). Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was column purified according to TotalPrep RNA Amplification Kit, and eluted in 60-80 µl of water. Quality of cRNA was controlled using the RNA Nano Chip Assay on an Agilent 2100 Bioanalyzer and spectrophotometrically quantified (NanoDrop).Nucleic Acid Extraction - Total RNA, from the transduced cells were isolated in phase lock tubes using the procedure of total RNA isolation with Trizol (Ambion) and the RNeasy Kit (QIAGEN). RNA 6000 Nano Lab on Chip kits and Agilent 2100 Bioanalyzer (Agilent-CA) were utilized to check RNA quality and quantity, respectively.Sample Collection - 786-O (CLS Cell Lines Service GmbH, Germany) was culture in RPMI-1640 medium , supplemented with 10% FCS and 1% Penicillin/Streptomycin.cells were transduced stably with shKDM5C or with shKDM5C+ KDM5C ORF to rescue the loss of KDM5C. Cells were injected s.c in mice and tumors reaching 100mm3 were treated with sunitinib (SU-11248, Pfizer, Inc., New York) at dose of 50mg/kg/day SID. Recovered tumors were snap frozen in liquid nitrogen. For transduction, Polybrene (Sigma-Aldrich) was used as a transduction reagent to infect cells with lentiviral particles. Cells were kept under the selection pressure of 2µg/ml Puromycin (Sigma-Aldrich) to ensure permanent presence of shRNA constructs. Kdm5c knockdown and overexpression vectors: KDM5C: TRCN0000234959 (Sigma-Aldrich®); shRNA control vector used was pLKO.1- SHC001 (Sigma-Aldrich®); ORF expression clone for KDM5C: 217EX-U1134-Lv152 (genecopoeia); Empty vector control for pReceiver-Lv152: 217EX-NEG-Lv152 (genecopoeia).Hybridization - Hybridization is performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed once in High Temp Wash buffer (Illumina Inc.) at 55°C and then twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes (in between washed with ethanol at room temperature). After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays are dried and scanned.E-MTAB-6745MIAME ScoreAssays and DataMAGE-TAB FilesArray DesignsChrisitan SchwagerAzadeh Fahim GolestanehfalseRole of KDM5C knock down in human renal cell carcinoma under Sunitinib treatmentHuman renal cell carcinoma cell line 786-O was stably transduced with shKDM5C or empty vector. Then KDM5C was rescued in shKDM5C and over expressed in cells with empty vector. The cells were used to produce tumors in nude mice and were treated with Sunitinib (SU-11248, Pfizer, Inc., New Yourk) at dose of 50mg/kg/day SID.2026-09-16T00:00:00Z2026-09-17T10:39:13.397Z2022-03-03T15:30:21.906ZE-MTAB-6748E-MTAB-6745E-MTAB-6747E-MTAB-6746E-MTAB-6749EFO_0002768EFO_0003814EFO_0002944EFO_0003813EFO_0005518EFO_0003815