|
| Status |
Public on Jul 04, 2011 |
| Title |
Fibroblasts not induced rep2 |
| Sample type |
RNA |
| |
|
| Source name |
E14,5 TH-GFP fibroblasts not infected
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype/variation: TH-GFP knock-in
developmental stage: E14.5
cell type: TH-GFP fibroblasts
infection status: non-infected
|
| Treatment protocol |
MEF cells were infected in MEF media, then 16–20 h in media containing lentivirus, the cells were switched into fresh MEF media containing doxycycline (2 mg/ml, Sigma) to activate expression of the transduced genes. After 48 h in MEF media with doxycycline the media was replaced with neuronal inducing media [(DMEM/F12 (Invitrogen), 25 μg/ml insulin (Sigma), 50 μg/ml transferrin (Sigma), 30 nM sodium selenite, 20 nM progesterone (Sigma), 100 nM putrescine (Sigma) and penicillin/streptomycin (Sigma)] containing doxycycline. The media was changed every 2–3 days for further 10 days.
|
| Growth protocol |
MEFs were isolated from TH-GFP knock-in mice embryos at E14.5 under a dissection microscope (Leica). The head, vertebral column (containing the spinal cord), dorsal root ganglia and all internal organs were removed and discarded to ensure the removal of all cells with neurogenic potential from the cultures. The remaining tissue was manually dissociated and incubated in 0.25% trypsin (Sigma) for 10–15 min to create a single cell suspension. The cells from each embryo were plated onto a 15-cm tissue culture dish in MEF media (Dulbecco’s Modified Eagle Medium; Invitrogen) containing 10% fetal bovine serum (FBS; Hyclone), b-mercaptoethanol (Sigma), non-essential amino acids, sodium pyruvate and penicillin/streptomycin (all from Invitrogen). In all experiments we used MEFs passaged no more than three times.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All samples were resuspended in Trizol, and extracted according to manufacturer's protocol. All samples were Dnase I-treated, and checked by Bioanalyzer 2100 for quality control.
|
| Label |
biotin
|
| Label protocol |
We used 10ng of total RNA with WT Ovation kit: Pico RNA amplification sys., Exon module, cDNA biotine module (according to NuGEN Technologies protocol).
|
| |
|
| Hybridization protocol |
22ng/mikL of fragmented and biotinilated cDNA was hybridizated for 16hours at 48°C on Affymetrix Mouse Gene 1.0 ST Arrays. We used FS0007 (labeling and washing script) protocol on Affymetrix Fluidic Station 450.
|
| Scan protocol |
Scanner 3000 7G (Affymetrix)
|
| Data processing |
Cel intensity values were computed using the Affymetrix Expression Console. Further data processing was performed in the R computing environment (http://www.r-project.org/) version 2.8.0 with BioConductor packages (http://www.bioconductor.org/). Robust Multi-Array Average (RMA) normalization was applied (Irizarry et al. 2003). Data were then filtered based on probe set intensity, so that only probesets that had intensity value >50 in at least half the arrays were retained. Statistical analysis was performed with limma (Smyth 2004). P-values were adjusted for multiple testing using Benjamini and Hochberg's method to control the false discovery rate (Hochberg and Benjamini 1990). Genes with adjusted P values below 0.01 were considered differentially expressed. Furthermore, a fold-change threshold cutoff was set to focus on genes whose expression level changes at least 2 times. Data were analyzed through DAVID Bioinformatics Resources v6.7 (Huang et al 2009, Dennis et al 2003).
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| |
|
| Submission date |
Feb 09, 2011 |
| Last update date |
Jul 04, 2011 |
| Contact name |
Paola Roncaglia |
| Organization name |
SISSA/ISAS (International School for Advanced Studies)
|
| Department |
Neurobiology
|
| Street address |
via Bonomea 265
|
| City |
Trieste |
| State/province |
TS |
| ZIP/Postal code |
34136 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE27174 |
Ascl1, Nurr1 and Lmx1 convert mouse and human fibroblasts into functional dopaminergic neurons without passing through an intermediate precursor state |
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