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Sample GSM384768 Query DataSets for GSM384768
Status Public on Mar 24, 2009
Title L2_H3K9Me3_3_of_3_rep_3
Sample type genomic
 
Channel 1
Source name ChIP, L2 larvae, L2_H3K9Me3_3_of_3_rep_3
Organism Drosophila melanogaster
Characteristics time point: L2
tissue: larvae
test: ChIP
antibody: H3K9me3
antibody manufacturer: Abcam
antibody catalog number: ab8898
antibody lot number: 422485
replicate: 3_of_3_rep_3
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 collection of second instar larvae (L2), new plates are added after a 2hours pre-egglaying at 9 am and flies are allowed to lay eggs for 24 hours. Plates are then kept at 25C for another 24 hours. 3. L2 larvae 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 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, L2 larvae, L2_H3K9Me3_3_of_3_rep_3
Organism Drosophila melanogaster
Characteristics reference: Input
time point: L2
tissue: larvae
replicate: 3_of_3_rep_3
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 collection of second instar larvae (L2), new plates are added after a 2hours pre-egglaying at 9 am and flies are allowed to lay eggs for 24 hours. Plates are then kept at 25C for another 24 hours. 3. L2 larvae 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 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 L2 larvae, L2_H3K9Me3_3_of_3_rep_3
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 20, 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 (3)
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
GSE18572 Discrete genomic domains exhibit co-occurrence of H3K9me3 and H3K27me3 histone modifications throughout Drosophila

Data table header descriptions
ID_REF
VALUE Normalized log ratios between the Cy3 channel and the Cy5 channel intensities.

Data table
ID_REF VALUE
125813 -0.62384735314969
211284 -0.322286580186362
116584 -0.250417514089992
3459 -0.0880834118847388
204749 0.712647548367365
36909 2.01883156043373
219799 -0.0132268503779729
115177 -0.58673932226587
191452 -0.559490027974647
80970 4.10872597973991
70521 4.05926852685585
173479 -0.747975918950449
114558 1.12051403987712
224367 -0.0367548610871663
30370 -0.604541424282107
11193 -0.0317972045219925
55621 1.35799538041596
23522 1.75948321683308
135143 2.54602631229184
241647 0.416169642658242

Total number of rows: 237869

Table truncated, full table size 5745 Kbytes.




Supplementary file Size Download File type/resource
GSM384768.txt.gz 67.4 Mb (ftp)(http) TXT
Processed data included within Sample table

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