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Sample GSM671658 Query DataSets for GSM671658
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).
 
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

Data table header descriptions
ID_REF
VALUE log2 RMA signal

Data table
ID_REF VALUE
10338001 13.06317
10338002 5.77111
10338003 11.42738
10338004 10.43201
10338005 1.65184
10338006 2.31079
10338007 1.65184
10338008 4.05514
10338009 10.03821
10338010 2.31079
10338011 6.42602
10338012 3.5185
10338013 2.52364
10338014 4.8203
10338015 2.72243
10338016 9.09536
10338017 13.48686
10338018 10.39993
10338019 5.77111
10338020 7.14451

Total number of rows: 35557

Table truncated, full table size 590 Kbytes.




Supplementary file Size Download File type/resource
GSM671658.CEL.gz 4.1 Mb (ftp)(http) CEL
Processed data included within Sample table

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