|
| Status |
Public on May 09, 2008 |
| Title |
N2A cells treated with empty vector, biological rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
N2A cells treated with empty vector
|
| Organism |
Mus musculus |
| Characteristics |
mouse N2A neuroblastoma cell line
|
| Treatment protocol |
Short hairpin knockdown of PTB in mouse N2A neuroblastoma cells was performed as described before (Boutz et al., 2007, Genes Dev 21:1636-1652). The efficiency of the PTB knockdown was monitored by western blot using PTB-NT primary antibody and Cy5 labeled secondary antibody (GE Life Sciences). The blots were imaged using Typhoon 9410 (GE Life Sciences). The band intensities were measured using ImageQuant and normalized to GAPDH. The GAPDH levels were monitored on the same western blot using mouse anti-GAPDH and Cy3 coupled secondary anti-mouse antibodys. In all cases the efficiency of the knockdown was close to 80% (data not shown).
|
| Growth protocol |
N2A cells were grown on DMEM suplemented with 10%FBS, glutamine, penicilin and streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was collected from adherent tissue culture cells using Trizol (Invitrogen) according to the manufacturer’s instructions. RNA samples were then treated with DNase I to remove residual DNA contamination and extracted with chloroform. RNA was quantified (A260) using a Nanodrop-1000 spectrophotometer (Nanodrop Technologies).
|
| Label |
biotin
|
| Label protocol |
Biotinylated sense-strand DNA for hybridization to Affymetrix exon arrays was prepared with the Affymetrix GeneChip whole transcript sense target labeling assay, using the standard 1 μg total RNA method according to the manufacturer’s protocols (Affymetrix, 900854). Ribosomal RNA reduction was performed using the RiboMinus Human/Mouse Transcriptome Isolation Kit (Invitrogen, K1550-01).
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| |
|
| Hybridization protocol |
The labeling assay yielded labeled single-stranded DNA that was fragmented and hybridized to Affymetrix Mouse Exon 1.0 ST Arrays (exon arrays) according to manufacturer’s protocols at the Stanford Protein and Nucleic Acid Facility.
|
| Scan protocol |
Hybridized Affymetrix Mouse Exon 1.0 ST Arrays (exon arrays) were scanned following the manufacturer’s protocols at the Stanford Protein and Nucleic Acid Facility.
|
| Description |
H1-3_MoEx-1_0-st-v1
|
| Data processing |
Raw image files were processed using Affymetrix GCOS 1.3 software to generate CEL data files. Exon array expression indexes were calculated using the GeneBASE program (Kapur et al., 2007, Genome Biol 8:R82). Briefly, the program employs a sequence-specific background model to correct background intensities of exon-array probes (Kapur et al., 2007, Genome Biol 8:R82), followed by an iterative probe selection procedure (Xing et al., 2006, PLoS ONE 1:e88) that selects a subset of highly correlated probes of a gene for expression index computation. Differential splicing events were detected using the MADS algorithm (Xing et al., 2008).
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| |
|
| Submission date |
May 05, 2008 |
| Last update date |
May 09, 2008 |
| Contact name |
Yi Xing |
| E-mail(s) |
yi-xing@uiowa.edu
|
| Phone |
319-384-3099
|
| Fax |
319-384-3150
|
| Organization name |
University of Iowa
|
| Department |
Internal Medicine, Biomedical Engineering
|
| Street address |
3294 CBRB, 285 Newton Rd
|
| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
| |
|
| Platform ID |
GPL6096 |
| Series (1) |
| GSE11344 |
MADS: a New and Improved Method for Analysis of Differential Alternative Splicing by Exon-tiling Microarrays |
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