NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM537564 Query DataSets for GSM537564
Status Public on Feb 18, 2011
Title atonalGFP+ t3 rep4
Sample type RNA
 
Source name Embryonic Drosophila cells isolated as atonal GFP+ by FACS at 7.75-8.75 hours after egg laying.
Organism Drosophila melanogaster
Characteristics genotype: atoGFP.7;w118
tissue: dechorinated embryo, flow sorted cells
developmental stage: 7.75-8.75 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

Data table header descriptions
ID_REF
VALUE log2 RMA/quantile normalized signal intensity

Data table
ID_REF VALUE
1616608_a_at 7.977903991
1622892_s_at 9.203115306
1622893_at 3.205626545
1622894_at 5.045618937
1622895_at 9.16348175
1622896_at 4.060663993
1622897_at 5.755660296
1622898_a_at 9.672494818
1622899_at 3.870944759
1622900_at 3.438481967
1622901_at 4.06553882
1622902_at 4.528463583
1622903_s_at 9.700043477
1622904_at 4.279350585
1622905_at 3.464872664
1622906_at 4.955657342
1622907_at 8.449736534
1622908_a_at 4.745391682
1622909_at 7.65016873
1622910_at 3.621028305

Total number of rows: 18952

Table truncated, full table size 432 Kbytes.




Supplementary file Size Download File type/resource
GSM537564.CEL.gz 2.1 Mb (ftp)(http) CEL
Processed data included within Sample table

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap