NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM700183 Query DataSets for GSM700183
Status Public on Mar 31, 2011
Title Snyder_EGL-5_GFP_L3_rep2 extraction2_seq2 aliquote 1
Sample type SRA
 
Source name Snyder_EGL-5_GFP_L3_rep2 extraction2_seq2 channel_1
Organism Caenorhabditis elegans
Characteristics strain: OP54(official name : OP54 genotype : unc-119(ed3) III; wgIs54 [unc-119(+) egl-5::TY1::EGFP::3xFLAG] 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 EGL-5::EGFP fusion protein is expressed in the correct egl-5 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the EGL-5 transcription factor. made_by : R. Waterston and S. Kim )
developmental stage: L3
genotype: unc-119(ed3) III; wgIs54 [unc-119(+) egl-5::TY1::EGFP::3xFLAG]
Sex: Hermaphrodite
Extracted molecule genomic DNA
Extraction protocol 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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer
 
Description channel ch1 is input DNA;
Data processing 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. Skip Illumina Data Merging protocol. Two biological replicates of ChIPed samples and one replicate of Input sample(total genomic DNA) were individually sequenced, and then the sequencing files from different biological replicates will be merged for Peak calling. Th sequencing file from one Input sample will serve as an input control for the PeakSeq base calling algorithm. Peak Calling 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 WS180
 
Submission date Mar 31, 2011
Last update date May 15, 2019
Contact name DCC modENCODE
E-mail(s) help@modencode.org
Phone 416-673-8579
Organization name Ontario Institute for Cancer Research
Lab modENCODE DCC
Street address MaRS Centre, South Tower, 101 College Street, Suite 800
City Toronto
State/province Ontario
ZIP/Postal code M5G 0A3
Country Canada
 
Platform ID GPL9309
Series (1)
GSE25796 Identification of Transcription Factor EGL-5::GFP Binding Regions in L3
Relations
SRA SRX054304
BioSample SAMN00254099

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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