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| Status |
Public on Nov 17, 2014 |
| Title |
SNPC-4 ChIP in staged L4s (5%) |
| Sample type |
SRA |
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| Source name |
staged L4 larvae from OP179 (SNPC-4-GFP transgenic strain)
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| Organism |
Caenorhabditis elegans |
| Characteristics |
developmental stage: L4
tissue: whole animal
chip'ped factor: SNPC-4
chip antibody: anti-GFP antibody (gift from Anthony Hyman)
strain: OP179
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| 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 at 20˚C except for YL457 and YL524 which were grown at 25˚C to enhance transgene expression. In addition, growth at 25˚C induced temperature-sensitive sterility for YL524 strain, which harbors glp-1(q224). Worms were harvested and washed free of bacteria in M9.
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| 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 either 1% (ChIP-seq data used in the tissue-specific analysis) or 5% (ChIP-seq data sets showing the developmental time course). 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 Anthony Hyman) or anti-Pol III antibody (gift from Jason Lieb).
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.
L1 animals were fed bacteria expressing RNAi clones from the Ahringer library (Fraser et al., 2000) until collection at the young adult stage. RNAi-treated young adults were harvested in TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was then treated with DNA-free DNAseI (Ambion, Austin, TX).
A total of 5 µg of DNAse-treated RNA harvested from snpc-4(RNAi) and wildtype young adult animals was subjected to the Illumina small RNA v1.5 sample preparation kit and 22-30 bp small RNAs were sequenced
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
ChIP was performed with anti-GFP of GFP-tagged SNPC-4
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| Data processing |
[ChIP-Seq] 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.
[RNA-Seq] Raw FASTQ files were processed and log2 reads-per-million (RPM) values were calculated for each sequence for normalization purposes. Reads were then aligned to known miRNA and 21U-RNA sequences extracted from C. elegans genome WS215, allowing for one mismatch.
Genome_build: [ChIP-Seq] WS220
Genome_build: [RNA-Seq] WS215
Supplementary_files_format_and_content: [ChIP-Seq] Bedgraph files were generated as an output of the SPP program
Supplementary_files_format_and_content: [RNA-Seq] Tab-delimited .txt files report abundance counts.
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| Submission date |
Dec 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Valerie Reinke |
| E-mail(s) |
valerie.reinke@yale.edu
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| Phone |
203-785-5228
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| Organization name |
Yale University School of Medicine
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| Department |
Genetics
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| Lab |
Reinke lab
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| Street address |
333 Cedar St
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06520 |
| Country |
USA |
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| Platform ID |
GPL9269 |
| Series (1) |
| GSE53412 |
The C. elegans SNAP complex component SNPC-4 coats piRNA domains and globally affects piRNA abundance |
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| Relations |
| BioSample |
SAMN02463669 |
| SRA |
SRX395521 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1291066_SNPC4_L4_IP_Rep0.density.scaled.bedgraph.gz |
13.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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