Single cell was plate into 96-well plate, on lethally irradiated 3T3-J2 cells (2.4 X 10^4/cm2) and cultured in 5% CO2 and humidified atmosphere in keratinocyte growth medium (KGM): DMEM and and Ham’s F12 media (3:1 mixture) containing Fetal Bovine Serum (FBS) (10%), insulin (5 mg/ml), adenine (0.18 mM), hydrocortisone (0.4 mg/ml), cholera toxin (CT, 0.1 nM), triiodothyronine (Liothyronine Sodium) (2 nM), epidermal growth factor (EGF, 10 ng/ml), glutamine (4 mM), and penicillin-streptomycin (50 lU/ml)
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cultured cells using the NucleoSpin ® RNA/Protein kit from Macherey-Nagel.
Label
biotin
Label protocol
Samples were amplified and labeled according to GeneChip 3' IVT PLUS Reagent Kit Protocol (Thermo Scientific).
Hybridization protocol
Samples were hybridized to GeneChip™ Human Genome U133 Plus 2.0 Array following the GeneChip 3' IVT PLUS Reagent Kit procedures (Thermo Scientific).
Scan protocol
Arrays were scanned following the GCS 3000 7G User guide (Thermo Scientific).
Data processing
Probe level signals were converted to expression values using the robust multi-array average procedure RMA (Irizarry et al., 2003) comprised in the Bioconductor affy package and a custom chip definition file based on the Entrez gene database (version 23, Dai et al., 2005). Microarray analyses were performed in R (version 3.5.0) using Bioconductor libraries and R statistical packages.
