strain: GOF18-EGFP age: day 5.5 tissue: Epiblast clone: GOF18
Treatment protocol
ESC were treated with the following reagents as indicated above: 100 units/ml LIF (Millipore, cat. ESG1107); 50 nM 4-hydroxytamoxifen (OHT) (Sigma, cat. H6278); Dkk1 100ng/ml (R&D, cat. 5897-DK); Sfrp1 100ng/ml (R&D, cat. 1384-SF).
Growth protocol
R1 mESC cells were cultured without feeders on plastic coated with 0.1% gelatin in the DMEM supplemented with 15% FCS (Hyclone Millipore, cat. ES-009-B), 100 mM 2‐mercaptoethanol (Sigma, cat. M7522), 1 × MEM non‐essential amino acids (Invitrogen, cat. 1140‐036), 2 mM l‐glutamine, 1 mM sodium pyruvate (both from Invitrogen). GOF18 EpiSCs were cultured in N2B27 medium supplemented with BSA 0,033%, Glutamax, b-Mercaptoethanol 0,1mM, bFGF 12ng/ml (Invitrogen, cat. 13256029) and Activin-A 20 ng/ml (R&D, cat. 338-AC). EpiSC were grown on fibronectin-coated plates and splitted every three days 1:20 by dissociation into small clumps with Collagenase Type IV 0,5 mg/ml (Gibco, cat. 17104-019).
Extracted molecule
total RNA
Extraction protocol
Total RNAs were isolated from cell cultures after 3 days growth in indicated conditions, using TRIzol® Reagent (Ambion, cat.15596-026), followed by clean up in accordance with the manifacturer's protocol provide with the kit.
Label
biotin
Label protocol
500 ng of each sample RNA were processed to generate biotinylated cRNAs following the Illumina TotalPrep RNA amplification Kit (Ambion, AMIL1791) protocol for Illumina Arrays.
Hybridization protocol
Standard Illumina hybridization protocol to MouseRef-8 v2 BeadChip Arrays (Illumina, 1128893), following the Direct Hybridization Assay guideline.
Scan protocol
BeadChip Arrays were scanned with a HiScan Array Scanner (Illumina) using the iScan Control Software (Illumina).
Description
replicate 2 SAMPLE 8
Data processing
The data were normalised using quantile normalization and background subtraction implemented by the GenomeStudio Gene Expression Module v1.0 Software (Illumina).
