Comment[ArrayExpressAccession] E-GEOD-46565 MAGE-TAB Version 1.1 Public Release Date 2014-04-29 Investigation Title Contribution of CBX4 to cumulus oophorus cell phenotype in mice, and attendant effects in cumulus cell cloned embryos Comment[Submitted Name] Contribution of CBX4 to cumulus oophorus cell phenotype in mice, and attendant effects in cumulus cell cloned embryos Experiment Description Cumulus oophorus cells play an essential role in oocyte development. CBX4 is a member of the Polycomb complex, which plays a role in regulating cellular differentiation. We used siRNA mediated knockdown of Cbx4 expression followed by Affymetrix array analysis to evaluate the role of CBX4 in cumulus cell differentiated state. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Midic Latham Hao Midic Garriga Person First Name Uros Keith Lanping Uros Judith Person Mid Initials E Person Email uros@msu.edu Person Affiliation Michigan State University Person Address Animal Sciences, Michigan State University, 474 S. Shaw Lane, East Lansing, MI, USA Person Roles submitter Protocol Name P-GSE46565-1 P-GSE46565-5 P-GSE46565-6 P-GSE46565-2 P-GSE46565-3 P-GSE46565-4 P-GSE46565-7 Protocol Description The data were preprocessed and analyzed with scripts written in R with Bioconductor libraries. Probeset summarization was done with Robust Multiarray Averaging (RMA), using a custom Chip Definition File (CDF) . Custom CDF was developed from the original Affymetrix CDF by removing probes that do not align to intended targets or cross-hybridize with non-homologous transcripts were removed from the respective probesets. Custom annotation were compiled by aligning remaining probe sequences to RefSeq mRNA sequences (downloaded 7/01/2013) for genes in GRC38 genome assembly. Significance Analysis of Microarrays (SAM) was used for identification of differentially expressed probesets. ID_REF = VALUE = RMA signal intensity (unlogged) Up to 50 ng of total RNA from each sample were subjected to two rounds of cDNA synthesis using the Arcturus RiboAmp HS Plus kit (Invitrogen). Labeled cRNA was produced using the Affymetrix GeneChip Expression 3M-bM-^@M-^Y Amplification for IVT Labeling Kit (Affymetrix, Santa Clara, CA). The biotin-labeled cRNA samples were fragmented and 10 M-5g were hybridized onto arrays. 12.5 ug of labeled cDNA was added to Affymetrix hybridization cocktails, heated at 99M-:C for 5 min and hybridized for 16 h at 45M-:C to MOE430V2 GeneChips (Affymetrix Inc., Santa Clara CA). The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain. Double-stranded siRNAs (21-mer) targeting mouse CBX4 were purchased from Qiagen (Valencia, CA). The corresponding target mRNA sequences for the siRNAs were: Cbx4-6 (S102731834): CAGGAAGAGCGG-CAAGT ATTA, Cbx4-7 (S104446673): AAGGTCCGAAGTTGAGGGAAA. Scrambled siRNA was used as a negative control. Cumulus cells were transfected with siRNA (25 nM Cbx4-6 and 25nM Cbx4-7) using lipofectamine RNAiMAX reagent (Invitrogen/Life Technologies, Grand Island, NY) according to the manufacturer protocol. After overnight culture, the medium was replaced and the cells were transfected using the forward transfection method. Thirty pmol of siRNA were diluted in 50 M-NM-