Comment[ArrayExpressAccession]	E-GEOD-26204
Public Release Date	2010-12-22
Investigation Title	Lieb SDQ4051_LEM2_N2_MXEMB							
Comment[Submitted Name]	Lieb SDQ4051_LEM2_N2_MXEMB
Experiment Description	modENCODE_submission_3166 This submission comes from a modENCODE project of Jason Lieb. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: The focus of our analysis will be elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus. 126 strategically selected targets include key histone modifications, histone variants, RNA polymerase II isoforms, dosage-compensation proteins, centromere components, homolog-pairing facilitators, recombination markers, and nuclear-envelope constituents. We will integrate information generated with existing knowledge on the biology of the targets, perform ChIP-chip analysis on mutant and RNAi extracts lacking selected target proteins, use extrachromosomal arrays to assess the ability of candidate identified sequence motifs to recruit targets in vivo, identify tissue-specific patterns of selected targets, and create integrated, quantitative models of transcription and whole-chromosome functions. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Strain: N2; Developmental Stage: Mixed Embryo; Genotype: wild type; Sex: mixed Male and Hermaphrodite population;  NUMBER OF REPLICATES: 1;  EXPERIMENTAL FACTORS: Developmental Stage Mixed Embryo; temp (temperature) 20 degree celsius; Antibody SDQ4051 LEM2 (target is LEM-2); Strain N2							
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Term Source Name	EFO
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Term Source File	http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl							
Person Last Name	modENCODE	Kohta	Lieb					
Person First Name	DCC	Ikegami	Jason					
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Person Email	help@modencode.org							
Person Affiliation	Ontario Institute for Cancer Research							
Person Phone	416-673-8579							
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Person Address	Ontario Institute for Cancer Research, MaRS Centre, South Tower, 101 College Street, Suite 800, Toronto, Ontario, Canada							
Person Roles	submitter							
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Protocol Name	P-GSE26204-7	P-GSE26204-6	P-GSE26204-2	P-GSE26204-4	P-GSE26204-3	P-GSE26204-1	P-GSE26204-5	P-GSE26204-8
Protocol Description	ChIP-chip_scanning_nimblegen_v2. Array scanning and raw data extraction were performed according to the protocol described in chapter 5 and 6 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, array signal was scanned by using a GenePix 4000B Scanner with associated software and saved as .tif files of the 532nm and 635nm images individually. Raw signal intensities of the images were extracted and saved as .pair files by using NimbleScan software v2.5 according to the NimbleScan User?s Guide.	ChIP-chip_label_hyb_nimblegen_v2. DNA was labeled and hybridized to NimbleGen C. elegans tiling array (HD2) according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008 with slight modifications. Briefly, amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.	Worm_embryo_extraction_v2. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl) + protease inhibitors (Calbiochem Cat# 539131). Using a Branson sonifier microtip, samples were sonicated on ice at the following settings: 35% amplitude, 0.9 sec on, 0.1 sec off, 12 pulses, 10 times. Cell debris was removed by centrifuging at 13,000 g for 15 minutes at 4ºC and taking the supernatant. Protein concentration was determined by Bradford Assay and extracts were aliquoted at stored at -80C.	Worm_LM-PCR_Amplification_for_ChIP-chip_vKI3 DNA was incubated with End Repair Enzyme mix (Epicentre, Madison) to ensure blunt ends and then with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends. The DNA fragments were ligated with T-over-hanged linker and then amplified by PCR with the longer linker oligonucleotide as a primer.	Worm_chromatin_immunoprecipitation_vKI3. Antibody was incubated with protein A/G beads at 4 oC for at least 1 hr and then washed by extraction buffer. For each reaction, 0.5-2 mg of protein extract was taken and sarkosyl was added to 1% final concentration. Before immunoprecipitation, 10% of extract was taken. The extract was mixed with the antibody-beads complex and incubated overnight at 4C. After washing beads, immune complexes were eluted from beads twice with 150uL elution buffer (1% SDS in TE with 250 mM NaCl) for 15minutes at 65C. Samples were treated with RNase A at room temperature for ? 1 hr then proteinase K at 55C for ? 1 hr. Samples were transferred to 65C overnight to reverse crosslinks. DNA was cleaned up Qiagen PCR purification kit. For a detailed protocol see http://www.modencode.org/	Worm_embryo_growth_and_harvest_v5. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Live embryos were cross-linked in M9 + 2% formaldehyde for 30 minutes at room temperature followed by quenching with 125mM glycine for 5 minutes. Embryos were then washed twice with M9 Buffer and once by FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl).  Pellets were frozen at -80C.	ChIP-chip_label_hyb_nimblegen_v2. DNA was labeled and hybridized to NimbleGen C. elegans tiling array (HD2) according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008 with slight modifications. Briefly, amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.	ChIP-chip normalization standard MA2C:JL:2 protocol. ChIP-chip_normalization_standard_MA2C_v2. First, all the IP and INPUT log ratio values are read from the pairdata file. Secondly, we build GC bins for INPUT and IP based on the GC counts for every probe sequence, which means that the INPUT or IP values for any probes which have the same GC counts will be put together. After that, for each GC bin, we calculate the mean for IP and INPUT data, and the covariance between this two channels. By default, the robust mean variance method is applied, which generalizes Tukey's theory of bi-weight estimation where the constant C is set to 2.  At last, we adjust the log ratio values for each probe by using the mean and covariance values for their corresponding GC bins, then these values are further normalized by their mean and standard derivation. In a single experiment, median within the sliding window defined by 2x bandwidth parameter is assigned as MA2C score at the center of each probe. In case of replicates, when we calculate the MA2Cscore afterwards, we take the median as the score from all the replicates for all the probes within the sliding window defined by 2x bandwidth parameter.  Processed data are obtained using following parameters: genome version is WS190
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Protocol Type	image_aquisition	hybridization	nucleic_acid_extraction	nucleic_acid_extraction	nucleic_acid_extraction	grow	labeling	feature_extraction
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Comment[SecondaryAccession]	GSE26204							
Comment[GEOLastUpdateDate]	2010-12-22
Comment[AEExperimentType]	ChIP-chip by tiling array							
Comment[GEOReleaseDate]	2010-12-22
Comment[ArrayExpressSubmissionDate]	2010-12-20
SDRF File	E-GEOD-26204.sdrf.txt