|
| Status |
Public on Jun 25, 2007 |
| Title |
Larval Pan-neural, biological rep 2 of set 2 |
| Sample type |
RNA |
| |
|
| Source name |
RNA obtained from all neurons after co-IP from F25B3.3::FLAG::PAB-1 transgenic
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
F25B3.3::FLAG::PAB-1 transgenic
|
| Treatment protocol |
For mRNA-tagging samples, synchronized L2 larvae were fixed in 0.5% formaldehyde, homogenized by passing through a French press (6000 psi on minicell) and using a Dounce homogenizer, and the cell-free extract isolated after a low-speed and high-speed spin. The FLAG-PAB bound RNA was isolated by immunoprecipitating with anti-FLAG antibodies, reversing the crosslink in a Tris buffer at 65 degrees Celcius, and TriZOL extracted.
|
| Growth protocol |
For MAPCeL analysis, gravid adults expressing F25B3.3::GFP (pan-neural) were subjected to bleach/NaOH treatment to release embryos. Embryos were harvested and then treated with chitinase to degrade the eggshell. Cells were dissociated and plated on poly-L-lysine coated dishes for 24 hrs to allow differentiation. Cells were removed from the culture dish and sorted using the FACStar Plus (Becton Dickinson, San Jose, CA) that had been flushed with egg buffer. ~90% enrichment of GFP+ cells was obtained. For mRNA-tagging, a synchronized population of larvae was obtained by the following method. Gravid adults expressing either F25B3.3::FLAG::PAB-1 (pan-neural) or unc-4::FLAG::PAB-1 (A-class) transgenes were subjected to bleach/NaOH treatment to release embryos. Embryos were harvested and allowed to hatch o/n in M9 liquid. Hatched L1 larvae were harvested and placed on plates containing food to restart development. After 22-24hr, when >80% of larvae had reached the mid-L2 stage, larvae were harvested for RNA extraction.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
For MAPCEL analysis, RNA was isolated from sorted cells using the micro-RNA isolation kit (Stratagene) using the recommended volumes for 1 million cells. For mRNA-tagging samples, following elution/crosslink, RNAs were isolated by TriZOL extraction and isopropanol precipitation (following manufacturer's protocol).
|
| Label |
biotin
|
| Label protocol |
A 2-round IVT protocol (modified from the Affymetrix small-sample protocol) was used to convert starting RNA into biotinylated aRNA.
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip C. elegans Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G.
|
| Description |
Gene expression data from all wildtype mid-L2 nematode neurons
|
| Data processing |
Data were normalized using Robust Multiarray analysis (RMA) in GeneTraffic (Stratagene). RMA normalized data were subjected to Significance Analysis of Microarray (SAM, Stanford) analysis. Each dataset was obtained with a False Discovery Rate (FDR) of less than or equal to 1%.
|
| |
|
| Submission date |
Jun 04, 2007 |
| Last update date |
Jun 25, 2007 |
| Contact name |
David Miller |
| E-mail(s) |
david.miller@vanderbilt.edu
|
| Phone |
6153433447
|
| Fax |
6159365673
|
| URL |
http://exploration.vanderbilt.edu/news/news_worm.htm
|
| Organization name |
Vanderbilt University
|
| Department |
Cell and Developmental Biology
|
| Street address |
465 21st Avenue South
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232-8240 |
| Country |
USA |
| |
|
| Platform ID |
GPL200 |
| Series (1) |
| GSE8004 |
Cell-specific microarray profiling of the C. elegans nervous system. |
|
