|
| Status |
Public on Dec 03, 2013 |
| Title |
PHA-4 ChIP input repA |
| Sample type |
SRA |
| |
|
| Source name |
embryonic stage
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
Stage: Fed L1
antibody: anti-GFP (polyclonal goat IgG; produced in Hyman lab)
|
| Growth protocol |
Synchronized L1 transgenic animals expressing NHR-25::GFP or PHA-4::GFP were fed on OP50 E. coli on agar plates for six hours and cross-linked with 2% formaldehyde for half an hour as described with modifications. The larvae were collected and crosslinked with 2% formaldehyde at room temperature for 30 minutes with gentle rotation. Glycine was added to a final concentration of 0.125 M for 5 minutes and the pellet was washed twice with 1 × PBS. The packed larvae pellet (equivalent to ~500,000 larvae) was resuspended in 0.4 ml nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) with proteinase inhibitor cocktail (Roche) for 10 minutes at 4 °C. The chromatin was then sheared in a 15-ml conical tube at 4 °C via a Diagenode Bioruptor (Diagenode, Denville, NJ) at the setting “H” for 2 cycles of 15 minutes (30s “ON”; 30s “OFF” for each cycle). The supernatant was collected and diluted with 9 parts of dilution buffer (50 mM Tris-HCl pH 8.0, 0.166 M NaCl, 1.1 % Triton X-100, 0.11 % sodium deoxycholate), supplemented with proteinase inhibitor cocktail (Roche).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The fixed worms were sheared with sonication and the cell lysate were used for ChIP with anti-GFP antibody
The libraries were constructed following Illumina standard protocol. Briefly, DNA was end-repaired (End-it DNA END-Repair kit, Epicentre) and the adenines were added to the 3’ ends by Klenow exo- (NEB). The barcoded adapters were ligated to the A-tailed fragments by T4 DNA ligase (NEB). The ligation products were purified by Agencourt AMPure XP system (Beckman Coulter, Inc) and PCR-amplified in a thermocycler for 16 cycles with KAPA HiFi hotstart mix (KAPA Biosystems) and PCR primers. Amplified libraries were size-selected on a 2% agarose gel and purified with MinElute columns (QIAGEN). The libraries were then sequenced on an Illumina Genome Analyzer IIx (Illumina, Hayward, CA) by the High-Throughput Genomics Unit (University of Washington) according to a standard protocol.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.7
ChIP-seq reads were aligned to the WS232 genome assembly using bowtie2 version 2.0.0-beta7 with the default parameters
Only uniquely mapped reads were remained for peak calling
peaks were called using MACS2 version 2.0.10 with the following setting: bw=200
Genome_build: WS232
Supplementary_files_format_and_content: bedGraph files were generated using MACS2; Scores represent normalized signal intensity.
|
| |
|
| Submission date |
Feb 27, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jiaofang Shao |
| E-mail(s) |
dreambetter@gmail.com
|
| Organization name |
McGovern Medical School, University of Texas Health Science Center
|
| Department |
Department of Biochemistry and Molecular Biology
|
| Lab |
Li Lab
|
| Street address |
6431 Fannin St
|
| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL13776 |
| Series (1) |
| GSE44710 |
Independent Regulation of Metabolism but Coordinated Control of Tissue Development by Epidermal Specific Transcription Factors in Caenorhabditis elegans |
|
| Relations |
| SRA |
SRX245917 |
| BioSample |
SAMN01931843 |
