NSC expansion medium, comprising NS-A media supplemented with N2 supplement, 10 ng/ml of each epidermal growth factor and basic fibroblast growth factor, 50 µg/ml bovine serum albumin, 1X penicillin/streptomycin/glutamine, and 1X nonessential amino acids
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol (Invitrogen Life Technologies, Carlsbad, USA), purified using RNeasy columns (Qiagen, Valencia, USA) according to the manufacturers’ protocol.
Label
biotin
Label protocol
Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit (Ambion, Austin, USA) to yield biotinylated cRNA according to the manufacturer’s instructions.
Hybridization protocol
750 ng of labeled cRNA samples were hybridized to each mouse-8 expression bead array for 16-18 h at 58°C, according to the manufacturer's instructions (Illumina, Inc., San Diego, USA). Detection of array signal was carried out using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) following the bead array manual.
Scan protocol
Arrays were scanned with an Illumina bead array Reader confocal scanner according to the manufacturer's instructions.
Description
Sample name: MEF.1
Data processing
Raw data were extracted using the software provided by the manufacturer(Illumina GenomeStudio v2009.2 (Gene Expression Module v1.5.4)). Array data were filtered by detection p-value < 0.05 (similar to signal to noise) in at least 50% samples (we applied a filtering criterion for data analysis; higher signal value was required to obtain a detection p-value < 0.05). Selected probe signal value was transformed by logarithm and normalized by quantile method. The comparative analysis between test group and control group was carried out using LPE test and fold-change. False discovery rate (FDR) was controlled by adjusting p value using Benjamini-Hochberg algorithm.
