Mouse ES, iPS and OKM cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) supplemented with 15% FBS (Gibco, Grand Island, NY), 100 μM beta-mercaptoethanol, 2 mM non-essential amino acid, 2 mM L-glutamine, 2 mM HEPES, and 10 ng/ml leukemia inhibitory factor (LIF) (Chemicon, Temecula, CA) on mitomycin C-treated primary MEF cells. MEF cells were cultured in DMEM+10% FBS
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted by SV Total RNA Isolation kit (Promega, Madison, WI) according to the protocol described in the kit. Total RNA quality was then determined by Agilent 2100 bioanalyzer (Agilent Technologies) according to manufacturer’s recommendations.
Label
Cy3
Label protocol
Immobilized, biotinylated aRNAs were detected by staining with cy3 Steptavidin (1ug cy3-SA per 1 ml of Illumina “Block E1”) for 10 minutes at room temperature.
Hybridization protocol
750ug of each aRNA in water (5ul) was suspended in Illumina “HYB” buffer (10ul) and heated to 65C for five minutes, then allowed to cool to room temperature. The samples were applied to Mouse8v2 Expression BeadChips and hybridized at 58C for 16-20 hours at high humidity. Arrays were washed according to Illumina standard protocol.
Scan protocol
Arrays were scanned on an Illumina BeadArray Reader. Laser power and PMT voltage were kept constant for Cy3 scans. Images were quantitated by Illumina Beadscan, v3.
Data processing
Quantitated data was imported into Illumina GenomeStudio software. On-slide spot replicates were averaged by GenomeStudio and individual spot probe was reported.
