|
| Status |
Public on Jul 21, 2013 |
| Title |
Class-IV da neuron replicate-2 |
| Sample type |
RNA |
| |
|
| Source name |
Class-IV da neuron-Larval L3 Stage
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain background: Oregon-R
genotype/variation: GAL4ppk.1.9, UAS-mcd8::GFP
developmental stage: Larval L3
tissue: Class-IV da neuron
|
| Treatment protocol |
Age-matched third larvae were used without any treatment.
|
| Growth protocol |
Drosophila stocks were raised on standard cornmeal-molasses-agar media at 25˚C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The isolation and purification of C-IV da neurons was performed as previously described (Iyer et al., 2009). Briefly, 40-50 age-matched third instar larvae expressing mCD8::GFP under the control of the GAL4ppk.1.9 or ppk-GAL80;GAL4221 driver were collected and washed several times in ddH20. The larvae were rinsed in RNAse away followed by ddH20 and finally dissected. The tissue was then dissociated using a combination of enzymatic and mechanical perturbations to yield single cell suspensions which were filtered using a 30µm membrane. The filtrate was incubated with magnetic beads coupled with rat anti-CD8 antibody (Invitrogen) for 60 minutes. Finally the C-I and C-IV neurons attached to the magnetic beads were separated using a powerful magnetic field. The cells were washed 5 times, before lysing them in SuperAmp™ (Miltenyi Biotec) RNA lysis buffer followed by storage of the RNA at -80˚C.
|
| Label |
cy3
|
| Label protocol |
250 ng of each of the cDNAs were used as template for Cy3 labeling which was performed according to Miltenyi Biotec’s undisclosed protocol.
|
| |
|
| Hybridization protocol |
The Cy3- labeled cDNAs were hybridized overnight (17 hours, 65°C) to an Agilent Whole Drosophila Melanogaster Genome Oligo Microarrays 4 x 44K (table 3) using Agilent’s recommended hybridization chamber and oven.Finally, the microarrays were washed once with 6x SSPE buffer containing 0.005% Nlauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies)
|
| Description |
Class-IV-Array-2
|
| Data processing |
The Agilent Feature Extraction Software (FES, v.10.5.1.1) was used to read out and process the microarray image files. The software determines feature intensities (including background subtraction), rejects outliers and calculates statistical confidences. GeneSpring (Agilent) was used to normalize and analyze raw dataset. The raw data was quantile normalized, and the values were thresholded to 1.
|
| |
|
| Submission date |
Apr 17, 2013 |
| Last update date |
Jul 22, 2013 |
| Contact name |
Daniel N Cox |
| E-mail(s) |
dcox5@gmu.edu, eramacha@masonlive.gmu.edu
|
| Phone |
703-993-4730
|
| Organization name |
George Mason University
|
| Department |
School of Systems Biology, Krasnow Institute
|
| Lab |
Cox Lab
|
| Street address |
4400 University Drive, MSN 2A1
|
| City |
Fairfax |
| State/province |
VA |
| ZIP/Postal code |
22030 |
| Country |
USA |
| |
|
| Platform ID |
GPL7300 |
| Series (1) |
| GSE46154 |
Microarray gene expression profiling of isolated class I and class IV Drosophila dendritic arborization sensory neurons |
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