HMT3522 T4-2 neoplastic breast epithelial cells derived from epidermal growth factor withdrawal (P. Briand et al., Cancer Res 56, 2039 (1996)).
Treatment protocol
T4-2 cells carrying pMFG-tet-GFP-NCOR2 and stably express GFP tagged NCOR2 by retrovirus-mediated gene transduction; treated with TRAIL (1 ug/ml) in three-dimensional reconstituted basement membrane culture for 4 hours.
Growth protocol
Cells were propagated in chemically defined H14 medium (V. M. Weaver et al., J. Cell Biol. 137, 231 (1997)) for 7 days, after which they were cultured on top of reconstituted basement membrane (Matrigel, BD Biosciences) for 6 days (G. Y. Lee et al., Nat Method 4, 359 (2007)).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from monolayer culture or 3D culture by a modified TRI reagent procedure (P. Chomczynski et al., Biotechniques 19, 942 (1995)) and purified using an RNeasy Mini Kit (Qiagen).
Label
biotin
Label protocol
Twenty microgram of total RNA from each sample was processed to produce biotinylated cRNA targets.
Hybridization protocol
Heat the following mixture at 99 degrees for 5 min: Control Oligonucleotide B2 (3nM) 5 ul, 20X Eukaryotic Hybridization controls 15 ul, Herring Sperm DNA (10mg/mL) 3 ul, BSA (50mg/ml) 3 ul, 2X Hybridization Buffer 150 ul, DMSO 30 ul, H2O to final volume of 300 ul. Moisten array with 200 ul 1X hybridization buffer and then place tough spots on and incubate in rotating oven for 10 minutes at 45 degrees. Transfer the cRNA hyb mix to the 45 degree oven to cool down for 5 minutes. Spin down tube at max speed in a microcentrifuge for 5 minutes. Remove the 1X hyb buffer from chips carefully and carefully load the spun-down samples (250 ul) onto chip. Reapply new tough spots. Place chips in rotating oven O/N.
Scan protocol
Arrays were scanned using the GeneChip Scanner 3000 (Affymetrix).
The hybridization intensity data was processed using the GeneChip Operating software (Affymetrix). Affymetrix .cel files (probe intensity files) were processed with ArrayAssist Lite (v3.4, Stratagene). The files were imported and processed with the GC-RMA algorithm to yield probe set intensities and additionally, Affymetrix Preset, Absent, Marginal flags were computed. These values were exported in .chp files, which were subsequently imported into the Partek Genomics Suite software (v6.2, Partek).
