ES cells were grown in standard ES media (15 0.000000CS, LIF, P/S, glutamine, non-essential amino acids, BME, DMEM high glucose) on irradiated MEFs. Cells were plated feeder free before RNA sample collection
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNAeasy microkit (Qiagen, Valencia, CA), DNAse treatment was performed on column
Label
Biotin
Label protocol
100 ng of total cellular RNA was double amplified and labeled with the Two-Cycle Target Labeling and Control Reagents kit P/N 900494 (Affymetrix Inc., Santa Clara, CA) following the manufacturers' protocol.
Hybridization protocol
Sample was hybridized to Affymetrix mouse 430 2.0 chips, washed, and scanned at the University of Minnesota Affymetrix Microarray Core Facility as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Protocol EukGE-WS2v5_450 .
Scan protocol
Array was scanned at the University of Minnesota Affymetrix Microarray Core Facility as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Pixel Size 1.56. Filter 570. Scanner ID 50209060. Number of Scans 1. Scanner Type M10
CEL files were loaded into GeneData Expressionist Refiner (GeneData, San Francisco, CA) to assess overall quality and obtain condensed single intensity values per probeset using the Microarray Analysis Suite Statistical algorithm (MAS 5.0). The mean of mean intensity values for all chips (192) was used to normalize the mean intensity of each chip. Normalization Target: 192, Tau: 0.015, Alpha1: 0.05, Alpha2: 0.065
