HFF were cultured in DMEM supplemented with 10% FBS, Glutamax(2mM). hiNRP and hNP cells were cultured in medium containing KnockOut™ DMEM/F-12 supplemented with StemPro® NSC SFM Supplement, Glutamax (2 mM), human bFGF (10 ng/ml) and EGF (10 ng/ml).
Extracted molecule
total RNA
Extraction protocol
Fibroblasts, hNPs and hiNRPs were collected in TRIzol (Invitrogen), followed by total RNA extraction as manual.
Label
Cy3
Label protocol
Double-strand cDNA (ds-cDNA) was synthesized from total RNA using an Invitrogen SuperScript ds-cDNA synthesis kit in the presence of 100 pmol oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc., USA). Briefly, ds-cDNA was incubated with 4 μg RNase A at 37°C for 10 min and cleaned using phenol:chloroform:isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a NanoDrop ND-1000. For Cy3 labeling of cDNA, the NimbleGen One-Color DNA labeling kit was used according to the manufacturer's guideline detailed in the Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA). One μg ds-cDNA was incubated for 10 min at 98°C with 1 OD of Cy3-9mer primer. Then, 100 pmol of deoxynucleoside triphosphates and 100U of the Klenow fragment (New England Biolabs, USA) were added and the mix was incubated at 37°C for 2 hours. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled ds-cDNA was purified by isopropanol / ethanol precipitation.
Hybridization protocol
Microarrays were hybridized at 42°C during 16 to 20 h with 4 μg of Cy3 labeled ds-cDNA in NimbleGen hybridization buffer/hybridization component A in a hybridization chamber (Hybridization System - NimbleGen Systems, Inc., Madison, WI, USA). Following hybridization, washing was performed using the NimbleGen Wash Buffer kit (NimbleGen Systems, Inc., Madison, WI, USA).
Scan protocol
After being washed in an ozone-free environment, Slides were scanned using an Agilent Scanner G2505C.
Description
This sample is the cells in neuronal restricted progenitor stage when human fibroblasts been reprogrammed by Sox2, c-myc, Brn4.
Data processing
Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and Gene level (*_RMA.calls) files were generated after normalization. All gene level files were imported into Agilent GeneSpring GX software (version 12.1) for further analysis. Differentially expressed genes between two samples were identified through Fold Change filtering. Hierarchical clustering was performed using the Agilent GeneSpring GX software (version 12.1). GO analysis and Pathway analysis were performed using the standard enrichment computation method.
Direct Conversion of Human Fibroblasts into Neuronal Restricted Progenitors
Data table header descriptions
ID_REF
VALUE
There are three steps of RMA: firstly, background subtraction was performed using a local background estimator. Secondly, quantile normalization (Bioinformatics 2003; 19:185) at probe level was done to make the expression distributions the same for all arrays. Last, probe set summarization was performed using Robust Multichip Average (RMA) algorithm as described by Irizarry, et al (Nucleic Acids Res. 2003; 31:e15 and Biostatistics 2003; 4:249).
