|
| Status |
Public on May 26, 2009 |
| Title |
Wild type embryo, 2-cell stage, Replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Cy3_WT mix embryos; Cy5_WT 2-cell single embryo
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain (cy3): N2
strain (cy5): N2
|
| Growth protocol |
2-cell embryos were collected from wild-type or mes-2 mothers and incubated at 20°C for 75 min. (2E), 120 min. (4E), 3h. (8E).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
cDNA from individual embryos was isolated as described (Robertson et al., 2004). Briefly, individual embryos were placed in 0.5 $B&L (Bl of cDNA 1st-strand buffer (Robertson et al., 2004), squashed with a small piece of coverslip and immediately frozen on dry ice. Upon thawing, 4 $B&L (Bl of 1st -strand buffer was added to each embryo. After heating to 65 °C (BC for 90 s, the tube was cooled to 45 °C (BC and reverse transcription initiated with the addition of 0.5 $B&L (Bl Superscript III. After 60 min, the reaction was terminated by heating to 70 °C (BC for 15 min, 0.5 $B&L (Bl RNAse H was added and RNA digested at 37 °C (BC for 20 min. The cDNA strands were polyA tailed by the addition of 5 $B&L (Bl of 2 $B!_ (B TdT buffer containing dATP, and terminal deoxynucleotidyl transferase, and incubated at 37 °C (BC for 15 min. Tailing was stopped by heating to 65 °C (BC for 10 min. 40 °C (Bl of PCR buffer was added to each tube followed by 50 cycles of PCR amplification. This primary RT-PCR amplification product could then be reamplified as needed to generate more total cDNA.
|
| Label |
Cy3,Cy5
|
| Label protocol |
PCR-amplified DNA samples (2-3 µg) derived from chromatin immunoprecipitation reactions were labeled using the Agilent Genomic DNA Labeling Kit PLUS. The labeling kit uses random primers and the exo-Klenow fragment to label DNA through incorporation of fluorescently labeled nucleotides (Cy3-dUTP or Cy5-dUTP). Following termination of the labeling reaction, fluorescently labeled DNA molecules were purified by isopropanol precipitation. Precipitated pellets were dried and then rehydrated in distilled water. The concentration of purified samples was determined using a NanoDrop ND-1000 spectrophotometer. A fraction of the labeled DNA (100 ng) was run on an Agilent BioAnalyzer to validate the size distribution of the labeled DNA molecules.
|
| |
|
| Hybridization protocol |
Fluorescently labeled cRNA samples (825 ng each) were fragmented and combined with Agilent Hi-RPM Hybridization Buffer. Microarray hybridizations were performed using Agilent SureHyb Hybridization chambers. Hybridization chambers were loaded onto a rotisserie in an Agilent Hybridization oven and were incubated at 65ºC for 17 hours with a rotational speed of 10 rpm. Following incubation, the microarray slide was washed for 1 minute each in Gene Expression Wash Buffer 1 (6X SSPE, 0.005% N-lauroylsarcosine; room temperature) and Gene Expression Wash Buffer (0.06X SSPE, 0.005% N-lauroylsarcosine; 31ºC) for 1 minute each. Microarray slides were briefly dipped in a solution of acetonitrile and dried.
|
| Scan protocol |
Microarray slides were scanned in an Agilent Technologies G2505B Microarray Scanner at 5 µm resolution. The scanner performs simultaneous detection of Cyanine-3 and Cyanine-5 signal on the hybridized slide. Data captured from the scanned microarray image is saved as a TIF file.
|
| Description |
total RNA from a single embryos
|
| Data processing |
TIF image files were processed with Agilent Feature Extraction Software version 9.5.1.
Non-uniform features were discarded and features with the same probe were averaged. Mean spot intensity was log (base 2) transformed and quantile normalized within groups. Group medians were adjusted to be equal.
|
| |
|
| Submission date |
Feb 20, 2009 |
| Last update date |
May 26, 2009 |
| Contact name |
Susan Mango |
| E-mail(s) |
susan.mango@hci.utah.edu
|
| Phone |
801-581-7976
|
| Fax |
801-585-6410
|
| Organization name |
University of Utah
|
| Department |
Oncological Sciences
|
| Lab |
Susan Mango
|
| Street address |
2000 Circle of Hope
|
| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
| |
|
| Platform ID |
GPL8209 |
| Series (1) |
| GSE14913 |
C. elegans embryos: control vs. mes-2 mutant |
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