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Status |
Public on Mar 03, 2016 |
Title |
Primary neurons |
Sample type |
RNA |
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Source name |
mouse embryonic primary neurons
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Organism |
Mus musculus |
Characteristics |
cell: primary neurons age: E13.5
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Treatment protocol |
The E13.5 ICR mouse embryos were employed to get the primary mouse embryonic fibroblasts (MEFs). MEFs were transduced with adenoviral supernatants in the presence of 4 mg/ml polybrene for 8 hours and then the clusters were refreshed by intermediate medium (low glucose with 5% FBS). And on the day 2, the same performance was repeated as that of day 1. And on the day3 and day5 after second infection, half-amount of the cell culture medium was switched to neuronal medium with 20 μM Forskolin (Sigma, F6886). After day 5, half cell culture medium was refreshed with neuronal medium every other day until the cells were ready for the sequent experiments. The cultures were incubated at 37 ºC in a humidified incubator with 5% CO2.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNAs were obtained from the indicated samples using TRIzol Reagent (Invitrogen, 15596-018) referring to the instruction manual. RNA was quantified using a NanoDrop-1000 spectrophotometer.
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Label |
Cy3
|
Label protocol |
Quick Amp Labeling Kit, One-Color (Agilent p/n 5190-0442).RNeasy Mini Kit (Qiagen p/n 74104) NanoDrop ND-1000
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Hybridization protocol |
Kit and Instruments:Agilent Gene Expression Hybridization Kit (Agilent p/n 5188-5242):10X Blocking Agent 25X Fragmentation Buffer. 2x GEx Hybridization Buffer HI-RPM. Hybridization Chamber, stainless (Agilent p/n G2534A). Hybridization Chamber gasket slides (Agilent p/n G2534-60003) Hybridization oven (Agilent p/n G2545A). Hybridization oven rotator for Agilent Microarray Hybridization Chambers (Agilent p/n G2530-60029)
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Scan protocol |
Instrument: Agilent Microarray Scanner (Agilent p/n G2565BA). Procedure: 1. Assemble the slides into an slide holder. 2. Place assembled slide holders into scanner carousel.3. Verify scan settings for one-color scans. Parameters Scan region Scan Area (61 x 21.6 mm) Scan resolution (μm) 5 5μm scanning mode Single Pass eXtended Dynamic range (selected) Dye channel Green Green PMT XDR Hi 100% XDR Lo 10% 4. Click Scan Slot m-n on the Scan Control main window where the letter m represents the Start slot where the first slide is located and the letter n represents the End slot where the last slide is located.
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Description |
Gene expression in primary neurons
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Data processing |
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, genes that at least 2 out of 6 samples have flags in Detected (“All Targets Value”) were chosen for further data analysis. Differentially expressed genes with statistical significance between the two groups were identified through Volcano Plot filtering. Differentially expressed genes between the two samples were identified through Fold Change filtering. Hierarchical Clustering was performed using the R scripts. GO analysis and Pathway analysis were performed in the standard enrichment computation method.
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Submission date |
Mar 02, 2016 |
Last update date |
Mar 03, 2016 |
Contact name |
Juan Zhang |
E-mail(s) |
zhangjuan@ioz.ac.cn
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Organization name |
Chinese Academy of Sciences
|
Department |
Institute of Zoology
|
Street address |
Beijing 100101, China
|
City |
Beijing |
ZIP/Postal code |
100101 |
Country |
China |
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Platform ID |
GPL11202 |
Series (1) |
GSE78841 |
Time course of gene expression signatures for Tet3 induced neurons |
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