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Sample GSM384815 Query DataSets for GSM384815
Status Public on Mar 24, 2009
Title E-20-24h_H3K27Me3_3_of_3_rep_3
Sample type genomic
 
Channel 1
Source name ChIP, embryos at 20-24 hours of development, E-20-24h_H3K27Me3_3_of_3_rep_3
Organism Drosophila melanogaster
Characteristics test: ChIP
tissue: embryos
time point: 20-24 hours of development
antibody: H3K27me3
antibody manufacturer: Upstate
antibody catalog number: 07-449
antibody lot number: DAM1387952
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 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 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 20-24 hours of development, E-20-24h_H3K27Me3_3_of_3_rep_3
Organism Drosophila melanogaster
Characteristics reference: input
tissue: embryos
time point: 20-24 hours of development
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 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 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 20-24 hours of development, E-20-24h_H3K27Me3_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
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
125813 -0.0724626007155853
211284 0.069303834653209
116584 0.328031258234789
3459 -0.429107060071414
204749 1.43788383965386
36909 1.63958155600002
219799 0.0912455772731021
115177 0.321356078966194
191452 0.505432935567031
80970 1.45630882135469
70521 0.491991100927071
173479 -0.76933733271055
114558 0.376817208153918
224367 5.10285483406124
30370 -0.756394070871243
11193 0.377966490413595
55621 5.72781474480891
23522 -0.281733261994491
135143 1.88968672426925
241647 5.67864007003063

Total number of rows: 237869

Table truncated, full table size 5742 Kbytes.




Supplementary file Size Download File type/resource
GSM384815.txt.gz 66.5 Mb (ftp)(http) TXT
Processed data included within Sample table

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