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Sample GSM729323

Query DataSets for GSM729323

Status

Public on May 24, 2011

Title

Snyder_CES-1_GFP_L4_rep2 extraction4_seq4 aliquote 1

Sample type

SRA

Source name

Snyder_CES-1_GFP_L4_rep2 extraction4_seq4 channel_1

Organism

Caenorhabditis elegans

Characteristics

strain: OP174(official name : OP174 genotype : unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119) 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 CES-1::EGFP fusion protein is expressed in the correct ces-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CES-1 transcription factor. made_by : S. Kim )
developmental stage: L4
genotype: unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119)
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 20ºC 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 ChIP DNA; Antibody information listed below: official name: Anti-eGFP;target name: eGFP;host: Goat;antigen: Full-length EGFP;clonal: Polyclonal;purified: Affinity;company: Lab;catalog: Hyman Goat anti-GFP;reference: PMID: 18391959;short description: A Bacterially expressed full-length EGFP protein tagged with 6xHis was used to raise a goat antisera and the resulting antibodies were affinity-purified.;

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. 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. 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

May 23, 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