|
| Status |
Public on Feb 18, 2011 |
| Title |
atonalmutGFP- t1 rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Embryonic Drosophila cells isolated as atonal GFP- from atonal mutant embryos by FACS at 5.75-6.75 hours after egg laying.
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: atoGFP.7;ato(mutant);w118
tissue: dechorinated embryo, flow sorted cells
developmental stage: 5.75-6.75 hours after egg laying
|
| Treatment protocol |
Embryos were dechorinated in 50% bleach for 2m30s.
|
| Growth protocol |
Eggs laid for 1 hour at 25C then embryos matured at 18C for either 11.5, 13.5 or 15.5 hours. Developmental times given as time after egg laying calculated as for development at 25C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The embryos were throughly washed in water then transferred to a dounce homogeniser that had been rinsed with S2 medium (Shields and Sang, Sigma #C3652, 5%FBS, Gibco #10500-056). As much water as possible was removed during transfer to the homogeniser using a Pastuer pipette. Embryos were then homogenised in 3ml of S2 medium with 25 gentle strokes of the loose pestle. The resulting cell suspensions were transferred to siliconised 1.5ml plastic tubes that had been rinsed with S2 medium. The rest of the protocol was carried out at room temperature. Cell suspensions were centrifuged at 1000g for 3 minutes, the supernatant was discarded, the pellet re-suspended in 1ml of protease solution (90% Trypsin-EDTA, Sigma #T4174, 10% 10xPBS) by gently pipetting up and down 10 times and was then incubated on a rotating wheel at room temperature for 7 minutes. Cells were pelleted by centrifugation at 1000g at 4C for 3 minutes, resuspended in 200ul S2, 1ml S2 was then added and they were then centrifuged again and resuspended in a final volume of 100um S2 ready for cell sorting. Cells were sorted into GFP+ and GFP- populations using a DakoCytomation MoFlo MLS flow cytometer. Typically between 300,000 GFP+ and 1,000,000 GFP- cells were collected from wild type embryos and 15,000-75,000 GFP+ and 500,000-1,000,000 GFP- cells were collected from mutant embryos. Cells were sorted into S2 medium and immediately spun down, homogenised in RNA extraction buffer (RLT) and snap frozen in liquid Nitrogen.
|
| Label |
biotin
|
| Label protocol |
Total RNA was isolated from frozen samples using the manufacturers recommended protocols using the RNeasy Mini Kit (Qiagen #74104). The quantity of total RNA was assessed using an Agilent 2100 Bioanalyzer and quantity by RT-PCR using a QuantiTect SYBR Green RT-PCR kit (Qiagen #204141) using the rpL32 gene as a control. Labeling was performed with a single round of amplification using the one-cycle target labeling kit (Affymetrix #900493) according to the manufacturers recommended protocols.
|
| |
|
| Hybridization protocol |
Hybridisation was performed with an Affymetrix Fluidic Station 400 according to the manufacturers recommended protocols.
|
| Scan protocol |
Scanning and quality control was performed with an Affymetrix Gene Array scanner 3000 7G according to the manufacturers recommended protocols.
|
| Data processing |
Quality control and normalisation of microarray expression data from raw CEL files was performed using the Bioconductor package AffyPLM (Gautier et al., 2004) using the standard RMA method with quantile normalisation.
|
| |
|
| Submission date |
Apr 26, 2010 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Ian Simpson |
| URL |
http://neuroregulatorygenomics.org
|
| Organization name |
University of Edinburgh
|
| Department |
Institute for Adaptive and Neural Computation
|
| Lab |
School of Informatics
|
| Street address |
10 Crichton Street
|
| City |
Edinburgh |
| ZIP/Postal code |
EH8 8AB |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE21520 |
The gene regulatory cascade linking proneural specification with differentiation in Drosophila sensory neurons |
|
| Relations |
| Reanalyzed by |
GSE119084 |
