Episomal vectors encoding reprogramming factors (OCT3/4, SOX2, KLF4, L-MYC, LIN28 and p53 shRNA) were transduced into fibroblasts on day 0 as described previously (Okita et al., 2011). The transfected cells were reseeded onto feeder layers on day 7, and maintained in embryonic stem cell medium (ReproCELL). Around day 30, the iPSC colonies were picked up and expanded. Cell culture was performed under 37°C , with 5% CO2 and 21% O2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted and purified using RNA mini kit (QIAGEN) according to manufacturer’s instruction.
Label
Cy3
Label protocol
100 ng of total RNA was first amplified with WT-OvationTM Pico System (Nugen). The RNA was labelled with Cy3 using Genomic DNA Enzymatic Labeling Kit (Agilent Technologies).
Hybridization protocol
Labelled cDNA was fragmented with Gene Expression Hybridization Kit (Agilent Technologies), followed by hybridization at 65˚C for 17 hrs.
Scan protocol
Images were acquired by DNA Microarray Scanner (Agilent Technologies)
Description
232_1_iPSC
Data processing
Raw data were processed with FeatureExtraction version 10.7.3.1. Normalization among samples were performed by GeneSpring GX 12.6.1. Signal were normalized to 75% percentile.
