|
| Status |
Public on Aug 22, 2009 |
| Title |
L3_PolII_2_of_3_rep_1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
L3_PolII
|
| Organism |
Drosophila melanogaster |
| Characteristics |
development stage: L3
antibody: PolII
antibody manufacturer: Covance
antibody catalog number: MMS-126R
antibody lot number: 14861301
test: ChIP
|
| Growth protocol |
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder. 2. For a collection of third instar larvae (L3), new plates are added after a 2 hours pre-egglaying at 9 am and flies are allowed to lay eggs for 24 hours. Plates are then kept at 25C for another 48 hours. 3. L3 larvae are collected at 8.45 am using a filter mesh and a brush. L3 larvae are the larvae not going through the first mesh. 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 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 |
L3_PolII
|
| Organism |
Drosophila melanogaster |
| Characteristics |
reference: Input
development stage: L3
|
| Growth protocol |
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder. 2. For a collection of third instar larvae (L3), new plates are added after a 2 hours pre-egglaying at 9 am and flies are allowed to lay eggs for 24 hours. Plates are then kept at 25C for another 48 hours. 3. L3 larvae are collected at 8.45 am using a filter mesh and a brush. L3 larvae are the larvae not going through the first mesh. 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 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 |
L3_PolII
|
| 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 |
Aug 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 |
GPL6950 |
| Series (2) |
| GSE15292 |
Drosophila at different time points of development: ChIP-chip, ChIP-seq, RNA-seq |
| GSE16702 |
ChIP-chip of PolII in Drosophila at different time points of development |
|
