NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM384797 Query DataSets for GSM384797
Status Public on Mar 24, 2009
Title E-16-20h_H3K27Me3_1_of_3_rep_1
Sample type genomic
 
Channel 1
Source name ChIP, embryos at 16-20 hours of development, E-16-20h_H3K27Me3_1_of_3_rep_1
Organism Drosophila melanogaster
Characteristics test: ChIP
tissue: embryos
time point: 16-20 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 16-20 hours egg laying, new plates are added after a 2hours pre-egglaying at 1 pm and flies are allowed to lay eggs for 4 hours. 3. Embryos are collected at 8.45 pm 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 16-20 hours of development, E-16-20h_H3K27Me3_1_of_3_rep_1
Organism Drosophila melanogaster
Characteristics reference: input
tissue: embryos
time point: 16-20 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 16-20 hours egg laying, new plates are added after a 2hours pre-egglaying at 1 pm and flies are allowed to lay eggs for 4 hours. 3. Embryos are collected at 8.45 pm 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 16-20 hours of development, E-16-20h_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 0.466582524728785
33716 0.559431570349055
157277 0.042285859085351
206658 -0.663084508270888
239126 -0.543167641891623
235304 1.30746688874979
134412 -0.330911180924683
53862 -0.550911180924683
198199 1.70807650717381
196182 -0.200243571921747
126974 -0.171714168786494
151252 1.62830177254939
192607 0.819813929063911
34113 -0.342656473100959
216057 2.28935672821917
135896 1.9276903729044
170628 0.0130209865368963
161522 -0.633561058767194
114968 0.330451048177484
62641 -0.298612982457479

Total number of rows: 237888

Table truncated, full table size 5736 Kbytes.




Supplementary file Size Download File type/resource
GSM384797.txt.gz 66.9 Mb (ftp)(http) TXT
Processed data included within Sample table

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap