Investigation Title RNAi profiling by array of human Ewings carcinoma cells to down regulate EWS-FLI1 Comment[Submitted Name] Genome-Wide Analysis of ETS Family DNA-Binding in vitro and in vivo Experimental Design cellular_modification_design in_vitro_design co-expression_design transcription profiling by array Experimental Design Term Source REF The MGED Ontology EFO Comment[AEExperimentType] transcription profiling by array Comment[AEMIAMESCORE] 5 Comment[SecondaryAccession] Comment[ArrayExpressReleaseDate] 2010-07-07 Comment[ArrayExpressAccession] E-MEXP-2759 Comment[MAGETAB TimeStamp_Version] 2010-08-10 14:14:17 Last Changed Rev: 13058 Experimental Factor Name RNAi Experimental Factor Type rnai Experimental Factor Term Source REF Person Last Name Wei Person First Name Gong-Hong Person Mid Initials Person Email gonghong.wei@helsinki.fi Person Phone +358(9)19125556 Person Fax Person Address "Genome-scale Biology Program, Haartmaninkatu 8, Helsinki, Helsinki, FIN-00014, Finland" Person Affiliation University of Helsinki Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-07-07 PubMed ID Publication DOI Publication Author List Publication Title Genome-Wide Analysis of ETS Family DNA-Binding in vitro and in vivo Publication Status Publication Status Term Source REF Experiment Description "We report here the DNA-binding profiles for all human and mouse ETS factors, which we generated using two different methods: a high-throughput microwell-based transcription factor DNA-binding specificity assay, and protein binding microarrays (PBMs). Both approaches reveal that the ETS binding profiles cluster into four distinct classes, and that all ETS factors linked to cancer, ERG, ETV1, ETV4 and FLI1, fall into just one of these classes. We identify amino acid residues that are critical for the differences in specificity between all the classes, and confirm the specificities in vivo using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for a member of each class. To determine whether the ChIP-seq peaks were near genes regulated by the respective ETS-factors, we used RNAi to downregulate EWS-FLI1 in SK-N-MC Ewing's sarcoma cells. The results indicate that even relatively small differences in in vitro binding specificity of a TF contribute to site selectivity in vivo." Protocol Name P-MTAB-14497 P-MTAB-14835 P-MTAB-14496 P-AFFY-2 Affymetrix:Protocol:Hybridization-Unknown P-AFFY-6 P-MTAB-14836 Protocol Type nucleic_acid_extraction specified_biomaterial_action grow labeling hybridization feature_extraction bioassay_data_transformation Protocol Description "Isolation of total RNA from SK-N-MC with different treatments was extracted by Qiagen RNeasy kit.
(Parameters: Extracted product = total_RNA, Amplification = none)" "SK-N-MC cells were grown in EMEM supplementing with penicillin/streptomycin and fetal bovine serum (FBS, 10%). For siRNA knockdown of EWS/FLI1 in SK-N-MC, the individual set of 4 siRNAs (Qiagen) against each gene were tested for knockdown efficiency by qRT-PCR, and two most effective single siRNA were used for further experiements (SI00387716 and SI00387730, Qiagen). The selected FLI1 siRNAs, or non-targeting control siRNA (Ctrl-control_1, SI03650325, Qiagen) were transfected into cells using HiPerFect Transfection Reagent (Cat. 301704, Qiagen). The final siRNA concentration was 10 nM. After 24h a second identical transfection was performed, and cells were harvested 48h later for RNA isolation. Prior to expression profiling, the efficiency of downregulation of the target gene and a set of known target genes were validated using real-time PCR." "SK-N-MC cells were grown in EMEM supplementing with penicillin/streptomycin and fetal bovine serum (FBS, 10%).
(Parameters: time unit = seconds, temperature unit = C)" Title: Affymetrix in vitro transcription. Description: Title: Affymetrix Generic Hybridization. Description: Title: Affymetrix CEL analysis. Description: "Expression profiling was performed using Affymetrix human genome U133plus2.0 arrays. The original CEL files of the experiments were imported into R (http://www.r-project.org/). The background was then corrected according to the GCRMA method. The probe sets were subsequently filtered for expression measurements to have a value of 100 fluorescence units in at least 25% of the samples. Differential expression analysis was performed by fitting a linear model as described in the Limma package; p values were adjusted according to Benjamini and Hochberg's method to control the false discovery rate. Probes showing significant differences (p < 0.01) between control samples were eliminated from the analysis. To facilitate comparisons between data sets and to increase statistical power, a relatively permissive criteria were used for inclusion of genes in further analyses; all genes with one probe set displaying absolute log fold change of more than 0.3 and adjusted p value of less than 0.01 in both experiments were included. The bioconductor package ""GOstats"" was used for gene ontology enrichment analyses.

Description in details of the table EWS-FLI1_RNAi_expression_profiling, which is the normalised data for this experiment in a tab-delimited text format. What were included are the log2 average expression values of the four separate experiments as columns.

The normalization process that led to these values was as follows:

1. Reading in the 8 CEL files of the experiments (the four separate experiments with two repeats each).

2. Applying gcRMA background correction.

3. Discarding probes with low expression across all samples (samples should have 100 fluorescence units in at least 25% of the samples (2 of 8 arrays).

4. Fitting a linear model to the expression levels using the Limma package.

Smyth, G. K. (2004). http://www.bepress.com/sagmb/vol3/iss1/art3

5. Discarding probes that show variance between the two control experiments (SKcon & SKneg) at a pVal of 0.01 as by the results from the limma analysis.

6. Average expression values from the expression sets were calculated for these probes and saved in the attached text file: EWS-FLI1_RNAi_expression_profiling." Protocol Parameters Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology mo The MGED Ontology SDRF File E-MEXP-2759.sdrf.txt Term Source Name ncbitax The MGED Ontology ArrayExpress The MGED Ontology EFO mo Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version