|
| Status |
Public on Feb 18, 2011 |
| Title |
Snyder_EGL-27_GFP_L1_rep1 extraction1_seq1 aliquote 1 |
| Sample type |
SRA |
| |
|
| Source name |
Snyder_EGL-27_GFP_L1_rep1 extraction1_seq1 channel_1
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: OP177(made_by : S Kim 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-27::EGFP fusion protein is expressed in the correct egl-27 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the EGL-27 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc119(ed3);wgIs177(egl-27::TY1 EGFP FLAG;unc119) official name : OP177 )
developmental stage: fed L1
genotype: unc119(ed3);wgIs177(egl-27::TY1 EGFP FLAG;unc119)
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.
In this ChIP Protocol whole C. elegans embryos are lysed and solubilized by sonication in the presence of protease inhibitors and non-ionic detergents. The cellular debris is removed by centrifugation and any formaldehyde cross-linked chromatin containing GFP epitopes is concentrated using an affinity-purified anti-GFP sera and Protein G-sepharose beads. After extensive washing, any immune complexes are eluted and the protein-DNA cross-links are reversed. The degree of sonication is assessed by running a small aliquot of DNA on an agarose gel. The chromatin IP is verified using quantitative PCR to assay if the experimental 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. This 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
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| |
|
| Submission date |
Feb 18, 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) |
| GSE25809 |
Identification of Transcription Factor EGL-27::GFP Binding Regions in L1 |
|
| Relations |
| SRA |
SRX044007 |
| BioSample |
SAMN00216272 |
