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Sample GSM384787 Query DataSets for GSM384787
Status Public on Mar 24, 2009
Title E-12-16h_H3K27Me3_1_of_3_rep_1
Sample type genomic
 
Channel 1
Source name ChIP, embryos at 12-16 hours of development, E-12-16h_H3K27Me3_1_of_3_rep_1
Organism Drosophila melanogaster
Characteristics test: ChIP
tissue: embryos
time point: 12-16 hours of development
antibody: H3K27me3
antibody manufacturer: Upstate
antibody catalog number: 07-449
antibody lot number: DAM1387952
replicate: 1_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 12-16 hours egg laying, new plates are added after a 2hours pre-egglaying at 5 pm 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 embryos 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 development, E-12-16h_H3K27Me3_1_of_3_rep_1
Organism Drosophila melanogaster
Characteristics reference: input
tissue: embryos
time point: 12-16 hours of development
replicate: 1_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 12-16 hours egg laying, new plates are added after a 2hours pre-egglaying at 5 pm 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 embryos 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 H3K27me3 in Drosohila embryos at 12-16 hours of development, E-12-16h_H3K27Me3_1_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 GPL6949
Series (3)
GSE15292 Drosophila at different time points of development: ChIP-chip, ChIP-seq, RNA-seq
GSE15423 ChIP-chip of H3K27me3 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
121180 0.610364122795255
33716 0.113006155679475
157277 -0.0814982398123045
206658 0.353307403888186
239126 -0.547770190001994
235304 2.96003944352475
134412 -0.244591427054921
53862 -0.2465244103547
198199 -0.111577605526378
196182 0.195461266006224
126974 -0.180324877682584
151252 0.163279328339956
192607 -0.204911242073807
34113 -0.614102298125994
216057 0.736988124150429
135896 -0.361424984710956
170628 1.08438636532318
161522 -0.155038600863547
114968 1.34370769765399
62641 -0.420756059879282

Total number of rows: 237888

Table truncated, full table size 5791 Kbytes.




Supplementary file Size Download File type/resource
GSM384787.txt.gz 23.5 Mb (ftp)(http) TXT
Processed data included within Sample table

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