|
| Status |
Public on Dec 29, 2015 |
| Title |
C. elegans WT rep4 |
| Sample type |
RNA |
| |
|
| Source name |
N2 wild-type strain
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: whole worm
developmental stage: L4 stage (synchronized)
genotype/variation: wild-type
|
| Treatment protocol |
No treatment. WT vs mutant comparison.
|
| Growth protocol |
We grew synchronized mid-L4 stage worms (wild-type N2 worms and cdk-8(tm1238) mutants that had been outcrossed 6 times to corresponding WT) that were grown on nematode growth medium (NGM)-lite agar plates seeded with Escherichia coli strain OP50, incubated at 20°C. 4 independent biological repeats (individual worms growths) were collected per strain. ~4000 worms/sample were harvested in M9, washed 3x15ml M9, flash-frozen in liquid N2 and then stored in a -80oC freezer until RNA processing
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by the Trizol extraction method as described in Taubert S et al., PLoS Genetics 2008 (PMID: 18454197). RNA quality was assessed on an Agilent 2100 Bioanalyzer using a Pico Chip (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified and labeled with Cy3-CTP using the Agilent low RNA input fluorescent linear amplification kits following the manufacturers protocol (Agilent). Labeled cRNA was assessed using the Nandrop ND-100 (Nanodrop Technologies, Inc., Wilmington DE), and equal amounts of Cy3 labeled target were hybridized to the microarrays (Agilent GPL10094: Agilent-020186 C. elegans (V2) Gene Expression Microarray (Feature Number version)).
|
| |
|
| Hybridization protocol |
Hybridizations were performed for 14 hrs, according to the manufacturers protocol.
|
| Scan protocol |
Arrays were scanned using the Agilent microarray scanner and raw signal intensities were extracted with Feature Extraction v9.1 software (Agilent).
|
| Description |
Gene expression in 4th larval stage
|
| Data processing |
This dataset was normalized using the quantile normalization method (Bolstad et al., 2003). No background subtraction was performed, and the median feature pixel intensity was used as the raw signal before normalization. A small number of probes (245) have replicate spots, and these were summarized by taking the median intensity. All arrays were of good quality and had similar foreground and background signal distributions for both mRNA and control probes. This suggests that quantile normalization is appropriate. To identify differentially expressed genes, a linear model was fit to the comparison to estimate the mean M values and calculate moderated t-statistic, B statistic, false discovery rate and p-value for each gene for the comparison of interest. Adjusted p-values (AdjP) were produced as described (Holm, 1979). All procedures were carried out using functions in the R package limma in Bioconductor (Gentleman et al., 2004; Smyth, 2004)
|
| |
|
| Submission date |
May 04, 2015 |
| Last update date |
Nov 23, 2022 |
| Contact name |
Stefan Taubert |
| Organization name |
UBC
|
| Department |
Medical Genetics
|
| Street address |
950 W 28th Ave, CMMT Room 2024
|
| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V5Z 4H4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL11346 |
| Series (2) |
| GSE68520 |
Genome wide expression analysis of Caenorhabditis elegans cdk-8(tm1238) null mutants (vs. WT N2 worms) |
| GSE220955 |
Genome wide expression analysis of Caenorhabditis elegans mdt-15(tm2182) null mutants (vs. WT N2 worms) |
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