|
| Status |
Public on Apr 09, 2011 |
| Title |
lin-54(n3423) dissected germline 2 |
| Sample type |
RNA |
| |
|
| Source name |
lin-54(n3423) mutant germline
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: germline
genotype: lin-54(n3423)
genetic background: N2
|
| Treatment protocol |
Embryos from young adults were harvested by the bleach-alkaline method, and filtered through 100 micron mesh. Germlines were dissected in 1X egg buffer to excise the germline including mitotic tip through meiotic late pachytene 24 hours after L4 stage.
|
| Growth protocol |
Strains for embryo or germline were cultured at 25o or 20oC, respectively, using standard methods.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
200 µL of embryo pellet was suspended in 1 mL of Tri reagent (Molecular Research Center, Inc. TR118), flash-frozen, and dounced. Total RNA was purified with RNAeasy mini kits (Qiagen), treated with DNase, and integrity examined on agarose gel. Germline RNA was isolated as described in Chi and Reinke (2006), and linearly amplified once using MessageAmp II aRNA Amplification Kit (Ambion).
|
| Label |
biotin
|
| Label protocol |
Fluorescence-labeled cDNA probes were prepared using the One-Cycle kit (Affymetrix) and the Enzo HighYield RNA Transcript Labeling Kit (Enzo) for embryo, and the 3' IVT Express Kit (Affymetrix) for germline
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| |
|
| Hybridization protocol |
cDNA probes of three replicates were hybridized to GeneChip C. elegans genome arrays (Affymetrix). Probe-preparation, hybridization, and scanning for DNA microarray were performed at the Genomics Core facility at University of Massachusetts Medical School.
|
| Scan protocol |
Array scanning was performed according to the manufacturer's instruction (Affymetrix)
|
| Data processing |
Statistical analyses were performed using R, a system for statistical computation and graphics ( http://www.r-project.org). The rma method in the affy package from Bioconductor was used in R to summarize probe level data and to normalize the dataset to remove across array variation. Log transformed data were used in subsequent analysis and plotting. WormBase version WS190 was used. To determine differentially expressed genes between wild-type and mutants, moderated T Statistics in limma was used with p-value ≤0.01, fold change ≥1.5.
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| |
|
| Submission date |
Apr 08, 2011 |
| Last update date |
Apr 09, 2011 |
| Contact name |
Kirsten Hagstrom |
| E-mail(s) |
kirsten.hagstrom@umassmed.edu
|
| Phone |
508-856-6843
|
| Fax |
508-856-8774
|
| Organization name |
University of Massachusetts, Medical School
|
| Department |
Molecular Medicine
|
| Lab |
Hagstrom Lab
|
| Street address |
377 Plantation street
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL200 |
| Series (2) |
| GSE28494 |
Germline and embryo gene expression of wild-type vs. mutants in lin-54, a component of the C. elegans DRM complex |
| GSE28853 |
Chromosome-biased binding and gene regulation by the C. elegans DRM complex |
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