KP cells were obtained from foreskin biopsies of healthy neonatal donors and expanded by cultivation onto lethally irradiated 3T3-J2 cells in growth FAD medium, a DMEM and Ham’s F12 media mixture (2:1) containing FCS (10%), penicillin-streptomycin (1%), glutamine (2%), insulin(5ug/ml), adenine (24.3ug/ml), hydrocortisone (0.4ug/ml), cholera toxin (50ug/ml), triiodotyronine (2nM). DKs were cultivated in EpiLife serum-free medium (Invitrogen) containing EpiLife defined growth supplements (HKGS, Invitrogen), penicillin-streptomycin (1%) and glutamine (2%).
Extracted molecule
total RNA
Extraction protocol
total RNA was extracted by RNeasy Mini Kit (QIAGEN) standard protocol and DNAse treated by RNase-Free DNase Set (QIAGEN)
Label
biotin
Label protocol
cRNA was biotinilated by IVT labeling kit standard protocol (Affymetrix)
Hybridization protocol
Biotinylated cRNA was hybridized onto GeneChip® HG-U133 Plus 2.0 Arrays according to the protocol supplied by the manufacturer (Affymetrix)
Scan protocol
standard Affymetrix protocol
Data processing
RMA of .CEL files using affy Bioconductor library. In RMA, PM values have been background adjusted, normalized using quantile normalization, and expression measure calculated using median polish summarization
