Comment[ArrayExpressAccession] E-GEOD-46775 MAGE-TAB Version 1.1 Public Release Date 2013-05-11 Investigation Title Snyder_UNC-39_GFP_EMB Comment[Submitted Name] Snyder_UNC-39_GFP_EMB Experiment Description modENCODE_submission_4635 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. 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-seq. BIOLOGICAL SOURCE: Strain: OP186(official name : OP186 genotype : unc119(ed3);wgIs186(unc39::TY1 EGFP FLAG C;unc119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The UNC-39::EGFP fusion protein is expressed in the correct unc-39 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the UNC-39 transcription factor. made_by : Unknown ); Developmental Stage: embryo; Genotype: unc119(ed3);wgIs186(unc39::TY1 EGFP FLAG C;unc119); Sex: mixed Male and Hermaphrodite population; EXPERIMENTAL FACTORS: Developmental Stage embryo; Target gene unc-39; Strain OP186(official name : OP186 genotype : unc119(ed3);wgIs186(unc39::TY1 EGFP FLAG C;unc119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The UNC-39::EGFP fusion protein is expressed in the correct unc-39 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the UNC-39 transcription factor. made_by : Unknown ); temp (temperature) 20 degree celsius Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name modENCODE Zhong Snyder Slightam Kim Murray Waterston Gerstein Niu Janette Raha Agarwal Reinke Sarov Hyman Person First Name DCC Mei Mike Cindie Stuart John Robert Mark Wei Judi Debasish Ashish Valerie Mihail Anthony Person Email help@modencode.org Person Affiliation Ontario Institute for Cancer Research Person Phone 416-673-8579 Person Address Ontario Institute for Cancer Research, MaRS Centre, South Tower, 101 College Street, Suite 800, Toronto, Ontario, Canada Person Roles submitter Protocol Name P-GSE46775-2 P-GSE46775-1 Protocol Description Illumina Data Analysis protocol. We used the recommended Illumina Data Analysis pipeline to process raw image files produced by the Genome Analyzer and generate aligned sequence reads. Illumina Data Merging protocol. This data analysis step effectively merges the processed data from each biological replicate (i.e., all of the high quality, unique, control ChIP-seq reads end up in one file, and all of the high quality, unique, experimental ChIP-seq reads end up in another file.These two files will become the input for the PeakSeq base calling algorithm. ChIP-seq replicate verification protocol. The PeakSeq method treats each aligned sequence read as a 200 nt fragment. The number of reads at each genomic site is counted, and compared to both a randomized model of the worm genome, and the number of parallel reads obtained from sequencing the input (non-ChIP) DNA. These calculations result in an enrichment ratio and a corresponding P-value. Processed data are obtained using following parameters: genome version is WS220 Worms are grown on peptone-enriched plates seeded with E. coli (HB101) and maintained according to standard protocol. Briefly, starved and synchronized L1s are first obtained, and then plated on peptone-enriched plates with HB101, which serves as a food source. Worms are grown at 20ÂșC to the desired developmental stage before harvesting. Since growth rates are frequently strain-specific, we use developmental milestones to determine developmental stages. Worms at the designed developmental stage are immediately crosslinked with 2% formaldehyde, quenched with 100 mM Tris buffer, washed with M9 buffer, and then stored at -80 as packed pellets. The current ChIP Protocol is modified from Ercan et al., Nature Genetics 39: 403-408 (2007). Worm samples are lysed and solubilized by sonication in the presence of protease inhibitors and non-ionic detergents. The cellular debris is removed by centrifugation and the supernatant containing any formaldehyde crosslinked chromatin is saved for preparing input DNA and collecting immunocomplexes. 10% (or 50 ul) of the above supernatant from each sample is saved as input sample (control). Input sample (also known as whole cell extract) represents the total genomic DNA and is processed later (treated with RNAase A, proteinase K and reverse crosslink steps) along with the ChIPed samples to isolate genomic DNA (input DNA). The remaining supernatant is incubated with affinity-purified anti-GFP antibodies and Protein G-sepharose beads to collect the immunocomplexes containing a targeted transcription factor that is C-terminally tagged with GFP and its genomic binding fragments. Alternatively, the 8WG16 mouse monoclonal antibodies and Protein A-sepharose beads are used to collect the immunocomplexes containing RNA polymerase II. After extensive washing, any immunocomplexes are eluted and the protein-DNA crosslinks are reversed. The degree of sonication is assessed by running a small aliquot of DNA on a 2% agarose gel. Quantitative PCR is used to check if the ChIPed sample contains any of the targeted transcription factor's known genomic binding sites. DNA fragments recovered following chromatin IP are size selected using gel electrophoresis. The DNA is then prepared for deep sequencing using the protocols and reagents provided by Illumina. This involves rendering the ends blunt, followed by the addition of single deoxy adenylate residues on each end. The fragments are ligated to Illumina's propietary adapters and amplified. After a final gel purification step the DNA is loaded into a flow cell for sequencing. Protocol Type normalization data transformation protocol nucleic acid library construction protocol Comment[SecondaryAccession] GSE46775 Comment[GEOReleaseDate] 2013-05-11 Comment[ArrayExpressSubmissionDate] 2013-05-09 Comment[GEOLastUpdateDate] 2013-05-13 Comment[AEExperimentType] ChIP-seq Comment[AdditionalFile:Data1] GSE46775_Snyder_UNC-39_GFP_EMB_GFP_combined.bedgraph Comment[AdditionalFile:Data2] GSE46775_Snyder_UNC-39_GFP_EMB_Input_combined.bedgraph Comment[AdditionalFile:Data3] GSE46775_Snyder_UNC-39_GFP_EMB_combined.GFF3 Comment[SecondaryAccession] SRP022320 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR849717-SRR849720 SDRF File E-GEOD-46775.sdrf.txt