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Sample GSM197737 Query DataSets for GSM197737
Status Public on Jun 25, 2007
Title Larval Reference, biological rep 3 of set 3
Sample type RNA
 
Source name RNA obtained from all cells by performing a control IP
Organism Caenorhabditis elegans
Characteristics Wildtype (N2 isolate) nematodes
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 cells
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.

Data table header descriptions
ID_REF
VALUE RMA

Data table
ID_REF VALUE
171720_x_at 200.9651107
171721_x_at 269.065047
171722_x_at 674.7143631
171723_x_at 73.91025797
171724_x_at 157.6346362
171725_x_at 6901.723132
171726_x_at 68.58740672
171727_x_at 18.4114391
171728_x_at 481.8012744
171729_x_at 396.1539359
171730_x_at 205.3095257
171731_x_at 377.981373
171732_x_at 264.5761581
171733_x_at 85.72076402
171734_x_at 5007.054563
171735_x_at 8023.053936
171736_x_at 157.1118836
171737_x_at 735.0020251
171738_x_at 976.6559648
171739_x_at 1050.281071

Total number of rows: 22625

Table truncated, full table size 497 Kbytes.




Supplementary file Size Download File type/resource
GSM197737.CEL.gz 3.3 Mb (ftp)(http) CEL
GSM197737.CHP.gz 5.8 Mb (ftp)(http) CHP
GSM197737.EXP.gz 355 b (ftp)(http) EXP
Processed data included within Sample table
Processed data provided as supplementary file

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