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| Status |
Public on Jun 01, 2013 |
| Title |
body_clk_ip_rep2 |
| Sample type |
SRA |
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| Source name |
body_clk_ip
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Clk
chip antibody: V5 (Sigma)
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| Growth protocol |
Drosophila flies were raised on standard food at 25 °C and under 12 hour light: 12 hour dark (LD) cycles.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, 5- to 10-days-old flies were collected 1h after lights-off (ZT13), frozen in liquid nitrogen, sieved to separate heads from bodies and kept in -80C until processed. 2 ml of fly heads or bodies were grinded in liquid nitrogen with a mortar pestle to a fine powder. Tissue powder was collected in a chilled 15 ml glass Dounce homogenizer (Wheaton) and immediately homogenized 30 times with a loose pestle in 50ml of NE Buffer (15mM HEPES pH 7.6, 10mM KCl, 0.1mM EDTA, 0.5mM EGTA, 350 mM sucrose, 0.1% Tween, 5mM MgCl2, 1mM DTT, 1mM PMSF plus Protease inhibitor cocktail (Roche)) with 1% formaldehyde (Sigma). Homogenization and fixation were done simultaneously to have a more precise snapshot of CLK and CYC binding. After the 30 strokes with the loose pestle, the fixing homogenate was poured to a 50ml Falcon tube and rotated for a total time of 10 min (starting from the addition of the NE Buffer). Fixation was quenched for 5 min at RT by the addition of 2.5 ml glycine (2.5M). Homogenate was filtered through a 60 um Streriflip filter (Millipore) and the nuclei were collected by centrifugation at 800g/5 min/40C. Nuclei pellet was washed twice in 10 ml cold NE Buffer (without formaldehyde) and twice more with cold RIPA Buffer (25 mM Tris-HCl 7.6, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40, 0.5mM DTT plus Protease inhibitor cocktail (Roche)). Nuclei were ressuspended in 2 ml cold RIPA Buffer and transferred to a 15ml falcon tube for sonication. Nuclei were sonicated on ice using an Omni Sonic Ruptor 250 Watts sonicator six times (heads) or four times (bodies) for 1min (Duty cycle: 30%, Output: 20%) each time, with 2 min intervals between the sonication steps. Sonicated chromatin was transferred to 1.5 ml eppendorfs and centrifuged at 10000g/10 min/40C. 15 ul of the supernatant were removed for the input sample while 1.5 ml of the supernatant were immediately incubated with 50 ul of blocked V5-agarose beads (Sigma) overnight at 4°C in a rotating wheel. After overnight incubation, beads were washed once with 1.4 ml Low Salt Buffer (20 mM Tris 8.1, 150 mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100), twice with 1.4 ml High Salt Buffer (20 mM Tris 8.1, 500 mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100), twice with 1.4 ml LiCl Buffer (10 mM Tris 8.1, 250 mM LiCl, 1mM EDTA, 1% sodium deoxycholate, 1% NP-40) and twice with 1.4 ml TE. Co-immunoprecipitated DNA fragments were eluted from the beads in 50 ul of V5 Elution Buffer (10mM HEPES, 1.5mM MgCl2, 0.25mM EDTA, 20% glycerol, 250mM KCl, 0.3% NP-40, 0.5mg/ml V5 peptide) three times consecutively, 30 min each, and the three elutes were combined (150 ul). The volumes of the V5 eluted chromatin and input were brought up to 500ul with IP Elution Buffer (10 mM Tris 7.5, 1 mM EDTA, 1% SDS, 100 mM NaHCO3, 200 mM NaCl) plus 15 ul of 10mg/ml RNase A, and incubated for 30 min 370C. RNA-digested chromatin was incubated overnight at 65C with Proteinase K, and in the following day the DNA was purified using standard Phenol:Chloroform extraction. Preparations of DNA libraries for single-end sequencing were done according to the illumina instructions with 36 cycles of extension. Up to 5ng ChIP or input DNA was used in each preparation.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Adult flies collected at ZT13
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| Data processing |
Base-calling was performed using RTA version 1.8.70.0
We mapped the reads to the Drosophila melanogaster genome reference dm3 (not chrU, chrUextra) obtained from UCSC using bowtie version 0.12.7, allowing only for uniquely mapping reads with up to 3 mismatches (parameters -m 1 -v 3 --best --strata).
We identified peak regions in ChIP samples compared to the corresponding input samples using peakzilla (http://www.starklab.org/data/peakzilla/). Confident peaks are selected with a score ≥ 10 and a fold enrichment of ChIP over input ≥ 2 and enriched regions with a score ≥ 2 and a fold enrichment of ChIP over input ≥ 2.
Genome_build: dm3
Supplementary_files_format_and_content: The peak files contain all enriched regions and are tab-delimited (1: chromosome, 2: start, 3: end, 4: summit, 5: score (IP-Input)*distribution score from peakzilla, 6: fold enrichment IP/Input; score and fold enrichment are normalized to 1 million reads per library)
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| Submission date |
Aug 29, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Anais Flore Bardet |
| Organization name |
IGBMC
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| Street address |
1 rue Laurent Fries
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| City |
Illkirch |
| ZIP/Postal code |
67404 |
| Country |
France |
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| Platform ID |
GPL11203 |
| Series (1) |
| GSE40467 |
cis-Regulatory Requirements for Tissue-Specific Programs of the Circadian Clock |
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| Relations |
| SRA |
SRX181433 |
| BioSample |
SAMN01141365 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM994692_body_clk_ip_rep2-body_clk_input_rep2_peaks.txt.gz |
18.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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