|
| Status |
Public on Mar 15, 2013 |
| Title |
genomic DNA of F2 SD, biological rep1 |
| Sample type |
genomic |
| |
|
| Source name |
Drosophila genomic DNA of F2 derived from yw and WE70 with abnormal heart phenotype (narrow heart tube).
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: F2 derived from yw and WE70
age: 1 week old female
|
| Treatment protocol |
1-week old female flies were anaesthetized with five minutes of FlyNap before semi-dissection. Whole body tissue are stored at -80c after semi-dissection.
|
| Growth protocol |
~15 female flies at 1 week old age are collected at room temperature (RT) on normal food source according to the different genotype.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each biological replicate genomic DNA was generated from 15 parental or selected F2 flies using DNA Easy prep kit (Qiagen). To avoid RNA contamination, genomic DNA was treated with 1.5μl RNAse at 37°C for 30 min. Linear amplification of genomic DNA was performed using the REPLI-g midi kit to a concentration of ~ 1μg/μl. Five μg of amplified genomic DNA was fragmented with 1 U RQ1 DNase (Promega) for 2 min at room temperature (18–22°C) in standard buffer followed by incubation with 2ul EDTA at 65°C for 10 min, and digestion was confirmed on 3% agarose gels by the existence of sheared products with ~50–100 bp length.
|
| Label |
biotin
|
| Label protocol |
50 μl fragmented genomic DNA was used in the labeling reaction with 22.5 U terminal deoxynucleotidyl transferase (rTDT) (Promega) and 1 μL (1 mM) Biotin N6-ddATP (Enzo) under the following temperature cycle: 37°C for 90 min, 99°C for 15 min and 12°C for 5 min. Gel-shift assays were performed to confirm labeling efficiency. Subsequently, the labeled products were hybridized to Affymetrix Chips (Drosophila Genome 2.0) using standard Affymetrix protocols at the Salk Institute’s Genomic Facility Center.
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| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of labeled genomic DNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using Affymetrix GeneChip scanner 3000 7G.
|
| Description |
Single Feature Polymorphism (SFP) data from genomic DNA pool of 15 F2 flies
Drosophila_F2_SD_1
|
| Data processing |
All statistical analysis code was written in R language. The required coordinate file for SFP analysis of Drosophila Genome 2.0 was obtained from Bioconductor. Briefly, the raw .CEL files from scanned arrays were read into R as the log-transferred intensity of 25mer perfect match (PM) probes after background correction and quantile normalization to ensure that all arrays have the same overall distribution. All 12 arrays were evaluated with >99% present call using MAS5 algorithm assuming high quality of hybridization.
Matrices contain log-transformed intensity of perfect match (PM) probes
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| |
|
| Submission date |
Mar 13, 2013 |
| Last update date |
Mar 15, 2013 |
| Contact name |
zhi zhang |
| E-mail(s) |
[email protected]
|
| Phone |
858-646-3100-3801
|
| Fax |
858-795-5298
|
| Organization name |
Sanford-Burnham Medical Research Institute
|
| Department |
Development and Ageing
|
| Lab |
Bodmer
|
| Street address |
10901 North Torrey Pines Rd.
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE45123 |
Single Feature Polymorphism (SFP) Data from Drosophila Genomic DNA |
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