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| Status |
Public on Aug 02, 2012 |
| Title |
OK107-H3K27me3 |
| Sample type |
SRA |
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| Source name |
Kenyon cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Head
chip antibody: H3K27me3 (Millipore: 07-449)
age: Adult
cell type: OK107
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Adult flies were anesthetized by CO2 and flash frozen in liquid N2. Heads were separated from abdominal-thoracic segments, wings and legs by vigorous vortexing followed by separation over dry ice cooled sieves. 600 - 10,000 frozen heads were added to 50mls of 10mM b-glycerophosphate pH7, 2mM MgCl2, 5mM sodium butyrate, 1X complete protease inhibitor cocktail (Roche: 11873580001) and the suspension was passed over a Yamato continuous flow homogenizer, set at 90-100 rpm, five to seven times. The homogenate was filtered over Miracloth (EMD Biosciences: 475855) and bought to 0.7mM b-mercaptoethanol and 0.5% NP-40. After six tractions in a 40ml Dounce homogenizer (pestle B), 600uls of antibody-adsorbed beads were added to 50mls of lysate. The binding reaction was run under continuous agitation at 4oC for 30 minutes. Beads were then collected on a magnet and washed three to four times in 50mls 10mM β-glycerophosphate pH7, 250mM sucrose, 2mM MgCl2, 25mM KCl, 5mM sodium butyrate. Bead bound nuclei in 20mls wash buffer were then passed over a 20um nylon mesh, returned to the magnet stand and resuspended in 1ml of 10mM β-glycerophosphate pH7, 250mM sucrose, 2mM MgCl2, 25mM KCl, 5mM sodium butyrate. Bead bound nuclei were collected on a magnet stand and resuspended in 2X500μls of 15mM Hepes pH7, 1mM KCl, 5mM MgCl2, 2mM CaCl2, 340mM sucrose, 0.5mM spermidine, 0.15mM spermine, 5mM sodium butyrate. Nuclei were then digested at 37oC after the addition of Micrococcal nuclease (Worthington: LS004798) to 0.025units/ul. The reaction was terminated by the addition of EGTA at 2mM. Nucleosomes were then extracted on ice for 30 minutes in 200-400μls of 15mM Hepes pH7, 200mM NaCl, 1mM KCl, 5mM MgCl2, 2mM EGTA, 340mM sucrose, 0.5mM spermidine, 0.15mM spermine, 5mM sodium butyrate. The extraction was repeated with the same buffer adjusted to 400mM NaCl. The supernatant from the second extraction was combined with the first and dialyzed for two hours at 4oC against 15mM Hepes ph7, 25mM KCl, 1mM β-mercaptoethanol, 1mM PMSF, 5mM sodium butyrate. Purified nucleosomes were then subjected to native-ChIP, and enriched DNA was end-repaired, linker-adapted and sequenced on an Illumina HiSeq 2000 to 50bp read length (Barski et al., 2007). The following antibodies were used to detect modified histones: H3K4me3 (Abcam: 8580), H3K27Ac (Abcam: 4729) and H3K27me3 (Millipore: 07-449).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
OK107-GAL4 (Adachi et al., 2003) driving unc84-GFP, primarily in Kenyon cells
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| Data processing |
Base calling performed by Illumina CASAVA 1.8
Reads were aligned to dm3 using BOWTIE v0.12.7 (bowtie -S -t -p 8 -m 1)
Coverage over all genomic positions counted with BEDTOOLS v2.15 (genomeCoverageBed -split -bg)
Coverage scaled to 10M total alignments. (custom Perl script)
Scaled coverage was converted to bigWig (wigToBigWig from ucsc)
Coverage over annotated genebodies, promoters (1kb centered around TSS), and all 10kb windows (scanned in 5kb increment across whole genome) was counted using BEDTOOLS v2.15 (coverageBed -counts -split -abam $BAM_FN -b $FEATUREBED)
Genome_build: dm3
Supplementary_files_format_and_content: bigWig tracks for visualization. supplemental file #1 (chipseq_perisoform_summary.txt) is a tab-delimited text file describing the modification levels across the genebody and promoter (1kb around TSS) in each sample. Supplemental file #2 (chipseq_perwindow_summary.txt) is a tab-delimited file containing H3K27ac, H3K27me3, and input signal in 10kb genomic windows for the OK107 and Tdc2 samples.
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| Submission date |
Apr 04, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Fred P Davis |
| E-mail(s) |
[email protected]
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| Phone |
571-209-4000-3437
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| Organization name |
HHMI Janelia Farm Research Campus
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| Street address |
19700 Helix Dr
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| City |
Ashburn |
| State/province |
VA |
| ZIP/Postal code |
20147 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (2) |
| GSE37032 |
Cell type-specific chromatin profiling of Drosophila neurons [ChIP-Seq] |
| GSE37033 |
Cell type-specific gene expression and chromatin profiling of Drosophila neurons |
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| Relations |
| SRA |
SRX135526 |
| BioSample |
SAMN00849460 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM909160_adult_OK107_brain_h3k27m3_111013_scaled10M.bw |
292.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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