biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicstranscription profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-6745We investigated the role of Kdm5c, Csf1 and Cd44 in LLC cell line. Cells were stably transduced with shRNAs against these genes. The expression profiles of these samples were compared to LLC cells transduced with empty vector control.biostudies-arrayexpressSample Collection - LLC cells (mouse Lewis Lung Carcinoma-ATCC® CRL-1642 ™) were cultured in DMEM media (ATCC) complemented with 10% fetal calf serum (FCS) (GIBCO). Stable knock down of individual genes was achieved by transducing cells with lentiviral particles, using Polybrene as tranduction reagent. 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).Scaning - Scanning and feature extraction were performed with Illumina BeadChip scanner (IsScan+) and Illumina feature extraction software.Nucleic Acid Extraction - Total RNA, from the transduced LLC 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.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).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-6746E-MTAB-6749E-MTAB-6748MIAME ScoreRaw DataAssays and DataorganisationMAGE-TAB FilesArray DesignsChrisitan SchwagerAzadeh Fahim GolestanehfalseRole of Kdm5c, Cd44 and Csf1 knock down in LLC cellsWe investigated the role of Kdm5c, Csf1 and Cd44 in LLC cell line. Cells were stably transduced with shRNAs against these genes. The expression profiles of these samples were compared to LLC cells transduced with empty vector control.2026-09-16T00:00:00Z2026-09-17T10:39:12.8Z2022-02-02T13:28:01.203ZE-MTAB-6745E-MTAB-6747E-MTAB-6746E-MTAB-6749E-MTAB-6748EFO_0002768EFO_0003814EFO_0002944EFO_0003813EFO_0005518EFO_0003815