|
| Status |
Public on Jan 11, 2010 |
| Title |
Dmel_adult_H3diMeK4_ChIP_female_rep2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
input, Drosophila melanogaster adult female
|
| Organism |
Drosophila melanogaster |
| Characteristics |
gender: female
reference: Input, 5-7 day aged adult flies
|
| Growth protocol |
Flies were grown at 25°C on standard cornmeal media. Wildtype flies for chromatin immunoprecipitaion were aged for 5-7 days post-eclosion, sexed and flash frozen.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
About 0.5 g of sex-sorted adult flies was cross-linked with 1.8% for 20 minutes at room temperature. After washing, the flies were completely homogenized and the cells were disrupted by cell lysis and nuclei lysis. The nuclear extraction was then sheared to 200-1000bp by sonication using a Misonix Sonicator 3000 and the soluble chromatin was collected for IPs. Specific antibodies were added to the chromatin extract for two hours and then were bound to protein A agarose beads at 4°C overnight. After washes and elution, the immunoprecipitated DNA was extracted and purified. DNA amplification was performed using a Ligation-mediated PCR (LM-PCR) protocol from FlyChip (http://www.flychip.org.uk/protocols/chip/lm_pcr.php).
|
| Label |
Cy3
|
| Label protocol |
600 ng of amplified DNA (ChIP enriched DNA or input DNA) were then labeled using the Cy3- or Cy5-labeled random nonamers (Trilink Biosciences, San Diego, USA) with Klenow enzymes.
|
| |
|
| Channel 2 |
| Source name |
H3diMeK4 ChIP, Drosophila melanogaster adult female
|
| Organism |
Drosophila melanogaster |
| Characteristics |
gender: female
test: ChIP, 5-7 day aged adult flies
antibody: H3diMeK4
|
| Growth protocol |
Flies were grown at 25°C on standard cornmeal media. Wildtype flies for chromatin immunoprecipitaion were aged for 5-7 days post-eclosion, sexed and flash frozen.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
About 0.5 g of sex-sorted adult flies was cross-linked with 1.8% for 20 minutes at room temperature. After washing, the flies were completely homogenized and the cells were disrupted by cell lysis and nuclei lysis. The nuclear extraction was then sheared to 200-1000bp by sonication using a Misonix Sonicator 3000 and the soluble chromatin was collected for IPs. Specific antibodies were added to the chromatin extract for two hours and then were bound to protein A agarose beads at 4°C overnight. After washes and elution, the immunoprecipitated DNA was extracted and purified. DNA amplification was performed using a Ligation-mediated PCR (LM-PCR) protocol from FlyChip (http://www.flychip.org.uk/protocols/chip/lm_pcr.php).
|
| Label |
Cy5
|
| Label protocol |
600 ng of amplified DNA (ChIP enriched DNA or input DNA) were then labeled using the Cy3- or Cy5-labeled random nonamers (Trilink Biosciences, San Diego, USA) with Klenow enzymes.
|
| |
|
| |
| Hybridization protocol |
Hybridizations of samples to the microarrays were performed at 60°C, followed by washes exactly as described (Parisi M, 2004, Genome Biol 5:R40).
|
| Scan protocol |
Arrays were scanned on an Axon GenePix 4000B (Molecular Devices Corporation, Sunnyvale, CA) and signal for each array elements were extracted with GenePix v.5.1 image acquisition software (Molecular Devices Corporation).
|
| Description |
ChIP-chip of H3diMeK4 in Drosophila adult female flies
|
| Data processing |
All microarray data were processed and analyzed in R/Bioconductor. The arrays were median centered (subtracting the weighted median from M-values for each array) and then normalized using quantile normalization based on the input channel as common reference across slides.
|
| |
|
| Submission date |
Apr 07, 2009 |
| Last update date |
Apr 25, 2012 |
| Contact name |
Brian Oliver |
| E-mail(s) |
[email protected]
|
| Phone |
301-204-9463
|
| Organization name |
NIDDK, NIH
|
| Department |
LBG
|
| Lab |
Developmental Genomics
|
| Street address |
50 South Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL20 |
| Series (1) |
| GSE15593 |
Sex-dependent and -independent X-chromosome histone modifications in Drosophila melanogaster |
|
