Triplicate samples (each 2 wells/sample) of each treatment were incubated at 4 degree with RNA-later (Sigma) overnight at each time point (day 0, day 2, day 4).
Growth protocol
E2f4f/f; R26-CreERT2 derived mTECs were cultured on transwells and treated with vehicle or 4-hydroxytamoxifen from day -5 to day 0.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from these samples (total 18) using a RNeasy Mini Kit (Qiagen, #74104) #74104).
Label
Biotin
Label protocol
Biotin labeling was performed using the Ambion WT Expression Kit (Life Technologies, Grand Island, NY) according to the manufacturer's protocol, followed by the GeneChip WT Terminal Labeling and Controls Kit (Affymetrix, Santa Clara, CA).
Hybridization protocol
The labeled, fragmented DNA was hybridized to the GeneChip Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA) for 18 hours in a GeneChip Hybridization oven 640 at 45oC with rotation (60 rpm). The hybridized samples were washed and stained using an Affymetrix fluidics station 450 with streptavidin-R-phycoerythrin (SAPE) and the signal was amplified using a biotinylated goat anti-streptavidin antibody followed by another SAPE staining (Hybridization, Washing and Staining Kit, Affymetrix, Santa Clara, CA).
Scan protocol
After staining, microarrays were immediately scanned using an Affymetrix GeneArray Scanner 3000 7G Plus (Affymetrix, Santa Clara, CA).
Raw Affymetrix CEL files were normalized to produce gene-level expression values using the implementation of the Robust Multiarray Average (RMA) in the affy Bioconductor package (version 1.36.1) and an Entrez Gene-specific probeset mapping (version 16.0.0) from the Molecular and Behavioral Neuroscience Institute (Brainarray) at the University of Michigan.
