|
| Status |
Public on Jan 11, 2010 |
| Title |
Drosophila_S2_mof-RNAi-treated_H3diMeK4_ChIP_rep1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Input, Drosophila melanogaster S2 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: S2 cells
sample type: input
|
| Growth protocol |
Drosophila S2 cells were grown at 25°C in Schneider's Drosophila Medium (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal Bovine serum (SAFC Biosciences, Lenexa, KS) and Penicillin-Streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
5-10 x 106 S2 cells with dsRNA or mock treatment were fixed with 1% formaldehyde in tissue culture media for 10 min at room temperature. After washing, 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 S2 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: S2 cells
antibody: H3diMeK4
|
| Growth protocol |
Drosophila S2 cells were grown at 25°C in Schneider's Drosophila Medium (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal Bovine serum (SAFC Biosciences, Lenexa, KS) and Penicillin-Streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
5-10 x 106 S2 cells with dsRNA or mock treatment were fixed with 1% formaldehyde in tissue culture media for 10 min at room temperature. After washing, 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 S2 mof-RNAi-treated cells
|
| Data processing |
All microarray data were processed and analyzed in R/Bioconductor. The arrays were normalized using quantile normalization based on the input channel as common reference across slides.
|
| |
|
| Submission date |
May 21, 2009 |
| Last update date |
Apr 25, 2012 |
| Contact name |
Brian Oliver |
| E-mail(s) |
briano@nih.gov
|
| 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) |
| GSE16344 |
Expression in Aneuploid Drosophila S2 Cells |
|
