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Sample GSM386110 Query DataSets for GSM386110
Status Public on Mar 28, 2009
Title E-20-24h_H3K4Me3_2_of_3_rep_2
Sample type genomic
 
Channel 1
Source name ChIP, embryos at 20-24 hours of development, E-20-24h_H3K4Me3_2_of_3_rep_2
Organism Drosophila melanogaster
Characteristics test: ChIP
antibody: H3K4me3
antibody manufacturer: Abcam
antibody catalog number: ab8580
antibody lot number: 411277
tissue: embryos
time point: 20-24 hours of development
replicate: 2_of_3_rep_2
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 20-24 hours egg laying, new plates are added after a 2 hours pre-egglaying at 9 am and flies are allowed to lay eggs for 4 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 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 20-24 hours of development, E-20-24h_H3K4Me3_2_of_3_rep_2
Organism Drosophila melanogaster
Characteristics reference: Input
tissue: embryos
time point: 20-24 hours of development
replicate: 2_of_3_rep_2
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 20-24 hours egg laying, new plates are added after a 2 hours pre-egglaying at 9 am and flies are allowed to lay eggs for 4 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 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 H3K4me3 in Drosophila embryos at 20-24 hours of development, E-20-24h_H3K4Me3_2_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 25, 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
GSE15424 ChIP-chip of H3K4me3 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
187694 -0.410903154553863
234359 0.637597604870733
25818 1.71261108998412
41947 -0.0519981655211327
205970 -0.252743115825983
130688 2.95096553424401
69684 0.23795016994785
171093 -0.642829427693062
101858 1.92197468368188
216572 -0.317654570768129
190489 -0.641003610973692
44898 -0.499488053097219
24398 4.50378368546831
116207 -0.567574431862542
137069 0.45180015343713
100722 0.0938527254536565
184904 0.603113437241726
100206 5.65174801405572
138224 -0.332751214238724
207216 -0.63936932997915

Total number of rows: 237894

Table truncated, full table size 5767 Kbytes.




Supplementary file Size Download File type/resource
GSM386110.txt.gz 66.8 Mb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data included within Sample table

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