|
| Status |
Public on May 29, 2009 |
| Title |
Pol_II_replicates 1,2,3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Embryo_E0-12
|
| Organism |
Drosophila melanogaster |
| Characteristics |
antibody: PolII
test: Chromatin
|
| Treatment protocol |
No treatment
|
| Growth protocol |
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder. 2. For a 0-12 hours egg laying, new plates are added after a 2hours pre-egglaying at 9 pm and flies are allowed to lay eggs for 12 hours. 3. Embryos are collected at 8.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 9 am in 1.8% formaldehyde at room temperature for 15 min.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Drosophila embryos are crushed in presence of 1.8% of formaldehyde for 15 minutes. After washing, the material is sonicated for 15 minutes to solubilize the chromatin. After centrifugation, the supernatant containing soluble chromatin is collected for IPs. Specific antibodies are added to the chromatin extract while one aliquot will be treated for DNA extraction corresponding to the reference samples. ChIP are carried overnight at room temperature. Protein A Sepharose beads are used to pull down the chromatin bound antibodies. After washes and finally elution, the DNA corresponding to the IP'd material is extracted and purified. Amplification of the DNA is done by linker-mediated PCR and used for labeling.
|
| Label |
biotin
|
| Label protocol |
Affymetrix Labeling Protocol (TdT labeling)
|
| |
|
| Channel 2 |
| Source name |
Embryo_E0-12
|
| Organism |
Drosophila melanogaster |
| Characteristics |
antibody: INPUT
|
| Treatment protocol |
No treatment
|
| Growth protocol |
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder. 2. For a 0-12 hours egg laying, new plates are added after a 2hours pre-egglaying at 9 pm and flies are allowed to lay eggs for 12 hours. 3. Embryos are collected at 8.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 9 am in 1.8% formaldehyde at room temperature for 15 min.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Drosophila embryos are crushed in presence of 1.8% of formaldehyde for 15 minutes. After washing, the material is sonicated for 15 minutes to solubilize the chromatin. After centrifugation, the supernatant containing soluble chromatin is collected for IPs. Specific antibodies are added to the chromatin extract while one aliquot will be treated for DNA extraction corresponding to the reference samples. ChIP are carried overnight at room temperature. Protein A Sepharose beads are used to pull down the chromatin bound antibodies. After washes and finally elution, the DNA corresponding to the IP'd material is extracted and purified. Amplification of the DNA is done by linker-mediated PCR and used for labeling.
|
| Label |
biotin
|
| Label protocol |
Affymetrix Labeling Protocol (TdT labeling)
|
| |
|
| |
| Hybridization protocol |
Affymetrix Hybridization Protocol - Hybridizations were performed at the Functional Genomics Facility (FGF) at the University of Chicago.
|
| Scan protocol |
Affymetrix Protocol - Scans were performed at the Functional Genomics Facility (FGF) at the University of Chicago.
|
| Description |
test CEL files: pol II.CEL Pol II 14.CEL Pol II 7.CEL
control CEL files: input 3.31.CEL Input 2.CEL Input 1.CEL
Pol_II_replicates 1,2,3
|
| Data processing |
.CEL files generated by the scanner are used as Input files in the MAT software to generate the signal files and peak finding
.bar and .bed are generated by MAT. .bar contains log2(IP/Input) fold changes for each probe. .bed files contains the peak calls at 1%fdr from MAT.
|
| |
|
| Submission date |
May 27, 2009 |
| Last update date |
Feb 02, 2015 |
| Contact name |
Kevin P. White |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Chicago
|
| Department |
Institute for Genomics and Systems Biology
|
| Street address |
900 E. 57th STR. 10th FL.
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60615 |
| Country |
USA |
| |
|
| Platform ID |
GPL6629 |
| Series (1) |
| GSE16245 |
Genomewide analysis of Insulator protein binding sites in Drosophila embryos at E0-12 |
|
| Relations |
| Named Annotation |
GSM409077_PolIIvsInputTripli_pvalue_Dm_BDGPv5.bed.gz |
