biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsThomas Fazziotranscription profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-31008To identify functional interactions among six chromatin regulators with key roles in embryonic stem cells, we examined gene expression changes in mouse ES cells with single and pair-wise combinations of knockdowns for those factors, along with control (EGFP) KDs. Effects on gene expression of RNAi-mediated knockdown of combinations of chromatin regulators. KDs were performed in duplicate for each combination for 48 hours.biostudies-arrayexpressSample Processing - In one well of a 24-well dish, 7X10^4 cells were transfected with 100 ng esiRNA using Lipofectamine 2000. After 12-15 hours, the media was replaced and cells were cultured until 48 hours post-transfection.Hybridization - Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.Labeling - Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.Growth Protocol - Feeder-free growth on gelatin coated dishes in standatd ES medium: KO DMEM + 10% FBS, glutamine, LIF, non-essential amino acids, beta-mercaptoethanol.Nucleic Acid Extraction - Trizol, followed by Invitrogen RNA micro columns.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsThomas FazzioImage Adquisition - Slides were scanned on the Agilent scanner immediately after washing.Assay Data Transformation - ID_REF = <br>VALUE = Log2 Normalized signal intensityFeature Extraction - The scanned images were analyzed with Feature Extraction Software 10.1 (Agilent) using default parameters. We used quantile normalization.falseA key role for Mbd3 in 5-hydroxymethylcytosine-dependent gene regulation in embryonic stem cellsTo identify functional interactions among six chromatin regulators with key roles in embryonic stem cells, we examined gene expression changes in mouse ES cells with single and pair-wise combinations of knockdowns for those factors, along with control (EGFP) KDs. Effects on gene expression of RNAi-mediated knockdown of combinations of chromatin regulators. KDs were performed in duplicate for each combination for 48 hours.2011-12-01T00:00:00Z2023-08-13T14:18:46.666Z2022-03-14T22:48:00.938ZE-GEOD-31008GSE31008EFO_0002768