In brief, the limbs and tails of neonatal mice were cut off, and the neonatal mouse skin was carefully peeled. The isolated neonatal mouse skin was then incubated in Collagenase type Ⅳ (2mg/ml, Gibco) for 2hrs at 37℃. After incubation, the epiderm was carefully removed from the surface of the skin. The remaining tissues were dissociated and minced into 1mm pieces, which were then resuspended by Dulbecco’s Modified Eagle Medium (DMEM, Hyclone), containing 10% Fetal bovine serum (Hyclone), 2x penicillin/streptomycin (Invitrogen). The isolated neonatal skin fibroblasts were collected by flowing through cell strainers.
Extracted molecule
total RNA
Extraction protocol
Total cellular RNA was extracted with TRIzol reagent (Sigma). Genomic DNA were removed with DNAase(Ambion), and 1ug RNA of each sample were used for reverse transcription with EasyScript Reverse Transcriptase (TransGen), according to the manufacturer's instructions. PCR amplification was performed using 2× EasyTaq SuperMix (TransGen).
Label
Cy5
Label protocol
Total mRNA from NBF1, NBF2, ICM1, ICM2, cm1, cm2 cells were labeled with Cy5.
Hybridization protocol
Hybridized to a mouse Oligo Microarray (Phalanx Mouse OneArray® v2, Phalanx Biotech) according to the manufacturer's protocol.
Scan protocol
After hybridization, arrays were scanned using GenePix 4000B scanner (Molecular Devices) and processed using the GenePix Pro 6.0 software (Molecular Devices).
Data processing
After removing control probes, a 14/33 presence call (SNR>=5 and foreground-background>0) was used to filter probes for the 33 microarrays, resulting in 12733 probes for further quantile normalization. The median of each sample was used for hierarchical clustering. Complete-linkage hierarchical clustering was performed using cluster 3.0 (written by Michael Eisen, Stanford University) with Spearman rank correlation coefficient as gene distances measurement and Pearson correlation coefficient as sample distances measurement.
