|
| Status |
Public on Mar 20, 2009 |
| Title |
E-12-16h_H3K9Me3_3_of_3_rep_2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ChIP, embryos at 12-16 hours of developments, E-12-16h_H3K9Me3_3_of_3_rep_2
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: embryos
time point: 12-16h
test: ChIP
antibody: H3K9me3
antibody manufacturer: Abcam
antibody catalog number: ab8898
antibody lot number: 422485
replicate: 3_of_3_rep_2
|
| Treatment protocol |
No treatment
|
| Growth protocol |
The sequenced Drosophila strain poupulation has been amplified and cultivated in population cages. The egg lay for collecting embryos is done in the cage with apple juice agar plates. Egg lay is done for 4 hours and embryos are collected 12 hours later for chromatin extraction.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Drosophila samples 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 |
Cy3
|
| Label protocol |
2 ug of amplified IP DNA are used for random primer labeling reaction (invitrogen). Cy3-dCTP is incorporated for the ChIP samples while Cy5-dCTP is incorporated for the Input chromatin sample. After washing of the labeled material on Centricon columns, 5ug of labeled DNA is used for hybridization.
|
| |
|
| Channel 2 |
| Source name |
Input, embryos at 12-16 hours of developments, E-12-16h_H3K9Me3_3_of_3_rep_2
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: embryos
time point: 12-16h
reference: Input
replicate: 3_of_3_rep_2
|
| Treatment protocol |
No treatment
|
| Growth protocol |
The sequenced Drosophila strain poupulation has been amplified and cultivated in population cages. The egg lay for collecting embryos is done in the cage with apple juice agar plates. Egg lay is done for 4 hours and embryos are collected 12 hours later for chromatin extraction.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Drosophila samples 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 |
Cy5
|
| Label protocol |
2 ug of amplified IP DNA are used for random primer labeling reaction (invitrogen). Cy3-dCTP is incorporated for the ChIP samples while Cy5-dCTP is incorporated for the Input chromatin sample. After washing of the labeled material on Centricon columns, 5ug of labeled DNA is used for hybridization.
|
| |
|
| |
| Hybridization protocol |
The hybridization solution is composed of 5 ug each of Cy3- and Cy5-labeled DNA, 50 ug of salmon sperm DNA, Agilent Blocking Agent and Agilent Hyb Buffer. The mixture is heated to 95*C for 3 min and then 37*C for 30 minutes immediately prior to hybridization. Samples are hybridized for 40 hours at 65*C. After hybridization the slides are washed in Agilent Wash Buffer 1 for 5 minutes at room temperature and then in Agilent Wash Buffer 2 for 5 minutes at 31*C.
|
| Scan protocol |
Channel 1 & 2 are for the Agilent arrays are scanned on Agilent's DNA Microarray Scanner G2505B at a resolution of 5 microns. Data is extracted from the images using Agilent's Feature Extraction Software 9.5.3.1.
|
| Description |
ChIP-chip of H3K9Me3 in Drosophila embryos at 12-16 hours of developments, E-12-16h_H3K9Me3_3_of_3_rep_2
|
| Data processing |
We preprocessed microarray data to normalize it and reduce experimental noise as described in Qi et al. (Qi et al., Nature biotechnology 2006). In short, the raw intensities from each channel was divided by the median intensity from that channel before computing a ratio to arrive at a median adjusted ratio. We further subtracted average value of negative control probes on the array and then added one as a regularization parameter.
|
| |
|
| Submission date |
Mar 19, 2009 |
| Last update date |
Aug 19, 2013 |
| Contact name |
Kevin P. White |
| E-mail(s) |
kpwhite@uchicago.edu
|
| 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 |
GPL6951 |
| Series (2) |
| GSE15292 |
Drosophila at different time points of development: ChIP-chip, ChIP-seq, RNA-seq |
| GSE15422 |
ChIP-chip of H3K9me3 in Drosophila at different time points of development |
|
