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Exon arrays of ENCODE tier 1, tier 2 and tier 3 cell types

ABSTRACT: This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Richard Sandstrom mailto:sull@u.washington.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). At cell harvest, a subset of cells was stored at -20oC in RNALater. Total RNA from 5 X 106 cells were purified using Ribopure (Ambion) according to vendor recommended protocols. Total RNA quality was assessed on RNA 6000 Nano Chips (Agilent) using a Bioanalyzer (Agilent). Approximately 3ug of total RNA for each cell type was sent to the Center for Array Technology (CAT) for labeling and hybridization to Affymetrix Human Exon 1.0 ST arrays. Briefly, a Whole Transcript Sense Target Labeling Assay (Affymetrix) was used by the CAT to reduce the rRNA, perform in vitro transcription (IVT) and create labeled sense strand DNA for hybridization to the arrays. Hybridizations were carried out according the manufacturers protocol. Intensity files from the scanned exon arrays were sent to our lab for analysis at the exon level (Affymetrix ExACT 1.2.1 software). Samples were quantile normalized with PM-GCBG background correction and PLIER (Probe Logarithmic Intensity Error) summarized. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

ORGANISM(S): Homo sapiens

PROVIDER: GSE19090 | GEO | 2009/11/19

REPOSITORIES: GEO

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Project description:To examine the effect of wtAPE1 and ubiquitin-APE1 fusion proteins on global gene expression. Total RNA from HEK293 derivatives that express vector alone (ctl), wt-APE1, or ubiquitin-APE1 fusion in the presence of doxycycline were analyzed. Three HEK293 derivatives expressing none (vector), wtAPE1, or Ub-APE1 fusion linked at APE1's 24th Lys were incubated with/without doxycycline (dox) 2 µg/ml for 16 hr. Each -/+ dox cultures were triplicated. Total RNA was extracted (Qiagen) and then analyzed by LSU's bioinformatics core. The quality and quantity of total RNA was assessed using an ND-1000 Spectrophometer (NanoDrop, Wilmington, DE), NanoChip assay and Bioanalyzer 2100 (Agilent). All samples were of high integrity with RIN # greater than 7 and A260/280 greater than 1.8. Procedures for cDNA synthesis, sense target labeling, and hybridization were carried out as described at http://media.affymetrix.com/support/technical/appnotes/wt_appnote.pdf (Affymetrix, Redwood City, CA). All experiments were performed using GeneChip Exon 1.0 ST Arrays and Whole Transcript Sense Target labeling Assay (version 4, Affymetrix). Overnight hybridization of fragmented single-stranded DNA was carried out in an Affymetrix GeneChip Hybridization Oven 640, then washed and stained with streptavidin-phycoerythrin using GeneChip 450 Microfluidics Station (Affymetrix). Chips were scanned with an Affymetrix High Resolution 3000 (G7) scanner. Signal and background intensities were quantitated by pixel intensity using Affymetrix GeneChip Operating Software (GCOS 1.4). Array quality control assessment was carried out using Robust Multi-chip Analysis (RMA) workflow for Core probesets in Expression Console (Affymetrix). Gene-level analysis for differential gene expression was analyzed in GeneSpring 7.3x (Agilent Technologies) using the sample CHP files. The RMA method was used for the background correction, normalization and average expression measures for the probesets. An expression filter (20th percentile cutoff) was applied to remove genes with low signal intensity values. For differential gene expression analysis, the Welch ANOVA test with asymptotic p-value computation was applied and transcripts with p-value (< 0.05) and fold change (2.0 fold) were selected.

Project description:To examine the effect of wtAPE1 and ubiquitin-APE1 fusion proteins on global gene expression. Total RNA from HEK293 derivatives that express vector alone (ctl), wt-APE1, or ubiquitin-APE1 fusion in the presence of doxycycline were analyzed. Three HEK293 derivatives expressing none (vector), wtAPE1, or Ub-APE1 fusion linked at APE1's 24th Lys were incubated with/without doxycycline (dox) 2 Âµg/ml for 16 hr. Each -/+ dox cultures were triplicated. Total RNA was extracted (Qiagen) and then analyzed by LSU's bioinformatics core. The quality and quantity of total RNA was assessed using an ND-1000 Spectrophometer (NanoDrop, Wilmington, DE), NanoChip assay and Bioanalyzer 2100 (Agilent). All samples were of high integrity with RIN # greater than 7 and A260/280 greater than 1.8. Procedures for cDNA synthesis, sense target labeling, and hybridization were carried out as described at http://media.affymetrix.com/support/technical/appnotes/wt_appnote.pdf (Affymetrix, Redwood City, CA). All experiments were performed using GeneChip Exon 1.0 ST Arrays and Whole Transcript Sense Target labeling Assay (version 4, Affymetrix). Overnight hybridization of fragmented single-stranded DNA was carried out in an Affymetrix GeneChip Hybridization Oven 640, then washed and stained with streptavidin-phycoerythrin using GeneChip 450 Microfluidics Station (Affymetrix). Chips were scanned with an Affymetrix High Resolution 3000 (G7) scanner. Signal and background intensities were quantitated by pixel intensity using Affymetrix GeneChip Operating Software (GCOS 1.4). Array quality control assessment was carried out using Robust Multi-chip Analysis (RMA) workflow for Core probesets in Expression Console (Affymetrix). Gene-level analysis for differential gene expression was analyzed in GeneSpring 7.3x (Agilent Technologies) using the sample CHP files. The RMA method was used for the background correction, normalization and average expression measures for the probesets. An expression filter (20th percentile cutoff) was applied to remove genes with low signal intensity values. For differential gene expression analysis, the Welch ANOVA test with asymptotic p-value computation was applied and transcripts with p-value (< 0.05) and fold change (2.0 fold) were selected. Three cell lines (HEK293/pcDNA-FRT empty vector, HEK293/pcDNA-FRT wtAPE1, HEK293/pcDNA-FRT ubAPE1), +/- 2Âµg/ml doxycycline for 16 h. Triplicates of each cell line/condition. Replicates were averaged.

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Exon arrays of ENCODE tier 1, tier 2 and tier 3 cell types

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