|
| Status |
Public on Dec 06, 2016 |
| Title |
LET-607 ChIP in staged YA rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
staged young adult (YA) from YL529 (LET-607-GFP transgenic strain)
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
developmental stage: YA
tissue: whole animal
chipped factor: LET-607
chip antibody: anti-GFP (gift from Kevin White)
strain: YL529
|
| Growth protocol |
Gravid adults were bleached and synchronized by L1 starvation. Starved L1s were plated on peptone-enriched NGM plates seeded with OP50, and grown until the desired developmental stage(young adult) at 20˚C. Worms were harvested and washed free of bacteria in M9.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For all ChIP-seq samples, worms were crosslinked in 2% formaldehyde in M9 buffer for 30 minutes rotating at room temperature. Crosslinking was quenched with 1M Tris pH 7.5, followed by two washes with M9, and one wash with FA buffer containing protease inhibitors. Final Triton X-100 concentration in all FA buffers used was 1%. Worm pellets were flash-frozen in liquid nitrogen and stored at -80˚C. Using the Fisher Scientific Sonic Dismembrator 550 (Pittsburgh, PA), samples were sonicated with a microtip on ice for sixteen cycles of 10 sec on, and 60 sec off on the “microtip” setting to fragment the majority of DNA within 200-800bp range. Sonicated extract containing 2mg of protein was immunoprecipitated as previously described (Zhong et al., 2010) with 7.5µg of anti-GFP antibody (gift from Kevin White).
ChIP-seq library preparation for enriched DNA and the genomic DNA input control for at least two biological replicates was performed as described in (Zhong et al. 2010), except Qiagen MinElute PCR purification and gel extraction kits were used after the Klenow step. Libraries were multiplexed as described in Lefrancois et al. (2009) in order to sequence four samples in a single flow cell using the Illumina Genome Analyzer II platform.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
ChIP was performed with anti-GFP of GFP-tagged LET-607
ENCFF281XQFrep2
processed data file: ENCFF489OCJread-depthnormsignal.bigWig
|
| Data processing |
FASTQ files were processed and mapped using BWA (Li et al., 2009), retaining only high quality alignments (Q ≥ 30).
Transcription factor binding sites were determined and scored using SPP peak caller (Kharchenko et al. 2008).
High confidence binding events were identified using IDR (Irreproducible Discovery Rate)
A cross-replicate threshold was obtained using a 5% IDR score threshold. Additionally, a rescue threshold method was also determined using IDR scoring. A final rank threshold for each data set was calculated by combining the cross-replicate and rescue threshold scores. Finally, reads from all replicates were pooled and the SPP and IDR framework was applied again to rank peaks by signal-score. The final rank threshold score determined from the individual replicates was used as a significance threshold for the ranked pooled-data peaks.
Genome_build: WS220
Supplementary_files_format_and_content: WIG files were generated as an output of the SPP program
|
| |
|
| Submission date |
Jul 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Valerie Reinke |
| E-mail(s) |
valerie.reinke@yale.edu
|
| Phone |
203-785-5228
|
| Organization name |
Yale University School of Medicine
|
| Department |
Genetics
|
| Lab |
Reinke lab
|
| Street address |
333 Cedar St
|
| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06520 |
| Country |
USA |
| |
|
| Platform ID |
GPL9269 |
| Series (1) |
| GSE84419 |
A novel small molecule that disrupts the oocyte-to-embryo transition in C. elegans |
|
| Relations |
| BioSample |
SAMN05392060 |
| SRA |
SRX1949397 |
