|
| Status |
Public on Jul 01, 2021 |
| Title |
soma-HSF1_depletion-NHS-HSF1_ChIP_replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
HSF-1 depletion from the soma, 20C (NHS)
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
treatment: non-heat-shock
age: young adults
tissue of tir1: soma
strain: JTL611 (hsf-1::aid::gfpI;ieSi57[eft-3p::TIR1::mRuby::unc-54_3'UTR+Cbr-unc-119(+)]II;unc-119(ed3)III)
chip antibody: GFP
|
| Treatment protocol |
Worms were transferred to either auxin (HSF-1 depletion) or ethanol (control) plates, and kept for 2 hours. Following this, worms were either directly collected or subjected to heat shock performed in a water bath pre-heated to 34°C. For heat shock, NGM plates were wrapped with parafilm and submerged for 30 min.
|
| Growth protocol |
Age synchronization was achieved by treatment of alkaline hypochlorite solution and eggs were hatched in M9. L1 larvae were transferred to 10 cm NGM plates seeded with OP50 and grown at 20°C to young adult stage.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Animals were collected from NGM plates, washed with M9 and crosslinked with 2% formaldehyde in PBS at room temperature for 15 min. Crosslinking was quenched by incubation with 0.1M Tris (pH 7.5) for 5 min. Worms were then washed three times in M9 and once in cold FA buffer (50 mM HEPES/KOH pH7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxyholate, 150 mM NaCl with Roche Complete protease inhibitors). Worms were then resuspend in 600 ul of FA buffer and lysed by douncing in a Kontes 2 ml glass dounce, and sonicated in Bioruptor to yield 200 bp–600 bp size DNA fragments. Immunoprecipitation (IP) was set up using 0.5 ug of chromatin, and either 5 ul of anti-full length GFP antibody (Clontech, living colors), or 3 ug of anti-RNA Pol II antibody (8WG16, BioLegend) in FA buffer (1 ml total volume), and incubated at 4 ºC overnight on a rotating wheel. The next day, 30 ul of protein-G Dynabeads were added to each IP.
ChIP-seq libraries were prepared using Accel-NGS 2S kit as per manufacturer’s instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
HSF-1_ChIP_peaks.xlsx
soma-depl-NHS-HSF1_combined_treat_pileup.bdg
Sample 11
|
| Data processing |
Sequencing reads were mapped to a non-repeat-masked version of the C. elegans WS235 genome using bowtie2 (Langmead and Salzberg, 2012) with the command bowtie2 --no-unal --very-sensitive –dovetail. Mapped reads in coordinated pairs, and with fragment size no bigger than 600bp were kept for downstream analyses. Duplicate reads were filtered for each replicate using MACS2 (Zhang et al., 2008) command macs2 filterdup -g ce --keep-dup auto.
HSF-1 peaks were called for each ChIP replicate of the control (no HSF-1 depletion) paired with the corresponding input sample using the command macs2 callpeak -g ce -f BEDPE --call-summits -q 1e-6. Common HSF-1 peaks in the biological duplicates were kept for subsequent analyses.
Filtered reads from biological replicates were combined to generate the bedgragh files using the command macs2 pileup -B. Given average fragment size of ChIP DNA is almost identical in all conditions for either HSF-1 or Pol II, pair-end reads were shifted half of the fragment length toward the center to generate the bedgraph files.
To determine the genomic regions with significant HSF-1 occupancy change by HSF-1 depletion, the bedgraph files in the control condition (EtOH) and the depleted condition (auxin) were compared using the command macs2 bdgdiff -l 180 -C 10.
To visualize and compare the ChIP-seq data in genome browser views, the bedgraph files were normalized to reads per million using MACS2 callpeak -B –SPMR.
Genome_build: WS235
Supplementary_files_format_and_content: The bedgraph files were generated with MACS2 for combined reads from two replicates and shown as reads per million (RPM).
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| |
|
| Submission date |
Nov 24, 2020 |
| Last update date |
Jul 01, 2021 |
| Contact name |
Jian Li |
| E-mail(s) |
[email protected], [email protected]
|
| Organization name |
New York Medical College
|
| Street address |
15 Dana Road
|
| City |
Valhalla |
| State/province |
NY |
| ZIP/Postal code |
10595 |
| Country |
USA |
| |
|
| Platform ID |
GPL26672 |
| Series (2) |
| GSE162063 |
Using Auxin-inducible-degradation System to Interrogate Tissue-specific Transcriptional Programs of HSF-1 in Reproduction and Heat Shock Response. [ChIP-Seq] |
| GSE162067 |
Using Auxin-inducible-degradation System to Interrogate Tissue-specific Transcriptional Programs of HSF-1 in Reproduction, Lifespan Assurance and Heat Shock Response. |
|
| Relations |
| BioSample |
SAMN16881579 |
| SRA |
SRX9567178 |
