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Sample GSM384766 Query DataSets for GSM384766
Status Public on Mar 24, 2009
Title L2_H3K9Me3_3_of_3_rep_1
Sample type genomic
 
Channel 1
Source name ChIP, L2 larvae, L2_H3K9Me3_3_of_3_rep_1
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_1
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_1
Organism Drosophila melanogaster
Characteristics reference: Input
time point: L2
tissue: larvae
replicate: 3_of_3_rep_1
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_1
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 (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

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 -0.77043775184811
211284 -0.426715710494166
116584 0.120946103664506
3459 0.0367818776437268
204749 0.514419166741715
36909 2.00014415280638
219799 -0.185237898475676
115177 -0.625892800436461
191452 -0.479476069738376
80970 3.4876552049726
70521 4.3469275777148
173479 -0.743171751243006
114558 1.84727479993702
224367 -0.0892563088568604
30370 -0.52040873180901
11193 -0.341920253864773
55621 0.740857715559412
23522 0.374446633898425
135143 2.61321395337618
241647 0.151117453206281

Total number of rows: 237869

Table truncated, full table size 5750 Kbytes.




Supplementary file Size Download File type/resource
GSM384766.txt.gz 67.5 Mb (ftp)(http) TXT
Processed data included within Sample table

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