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| Status |
Public on Sep 12, 2013 |
| Title |
Dmel_vHB |
| Sample type |
SRA |
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| Source name |
Blastoderm embryos
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| Organisms |
Drosophila melanogaster; Drosophila pseudoobscura; Drosophila virilis |
| Characteristics |
developmental stage: Blastoderm
strains: Oregon R, MV2-25, V46
chip antibody: HB, counter-purified agians D. virilis protein (Li et al., 2008)
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| Growth protocol |
Embryos collected from large population cages for 1 hour, aged to appropriate stage, fixed in formaldehyde.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was isolated by CsCl gradient ultracentrifugation, fragmented and immunoprecipitated with affnity-purified antibody
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly DNA fragments were converted to phosphorylated blunt ends using T4 DNA polymerase, Klenow DNA polymerase, and T4 polymerase kinase, a 3' A base overhang was added using Klenow DNA polymerase exo- (3' to 5' exo minus), and Illumina adapters were ligated to the fragments. We carried out the PCR step for enrichment of adapter-modified DNA prior to the library size selection, and limited the amplification to 15 cycles. After the amplification step, we size-selected DNA fragments of 150-250 bp.
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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 |
Similar quantities of chromatin from the three species D. melanogaster, D. pseudoobscura and D. virilis were pooled after sonication and before ChIP. ChIP product was sequenced and reads were mapped to each species separately and reads that ambiguously mapped to several genomes were tossed. A very small quantity of mapped reads (less than 1%) were discarded, and the pooling had virtually no effect on read mapping.
ChIP_metatable_HB.txt
HB: Inter species comparison of peak location, alignment quality, peak height, peak height based on motif content.
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| Data processing |
Basecalls performed using CASAVA version 1.4
We used the Apr. 2006 assembly (Flybase Release 5) of the D. melanogaster genome, the February 2006 assembly (Flybase release 2) of the D.pseudoobscura genome and the February 2006 assembly (Flybase release 1) of the D.virilis genome. We trimmed all sequenced tags so that their average quality was above 30 and mapped the tags to the genomes using Bowtie v0.12.7 {Langmead:2009fv} with command-line options ‘-v 1 -m 1’ for small reads (length below 35bp) and ‘-v 1 –m 3 for long reads (length above 70bp), thereby keeping only tags that mapped uniquely to the genome with at most one or three mismatch. ChIP data was parsed independently for each experiment using two separate peak callers. First, we used MACS (version 1.4) {Zhang:2008gm}, with the following parameter “-g dm --off-auto --nomodel --pvalue=1e-2” and “--shiftsize=110 --mfold=10,10000 --slocal=2000 --llocal=20000"" or ” --shiftsize=60 --mfold=4,10000"" depending on the length distribution for DNA fragment sizes prior to sequencing. We also called peaks using Grizzly Peak fitting program (Capaldi et al., 2008, Harrison et al., 2011) with estimated DNA fragment length of 150 or 250.
We then intersected the two sets of peaks, and filtered out all peaks not supported by both methods. To account for low complexity peaks and possible PCR artifacts, we further removed peaks with negative correlation (<-0.1) among the Forward and Reverse reads, peaks where 60% of the reads mapped to less than 1% of the positions, and peaks whose height was less than three times the height of Input reads in the same locus.
ChIP data was parsed independently for each experiment using two separate peak callers. First, we used MACS (version 1.4) {Zhang:2008gm}, with the following parameter “-g dm --off-auto --nomodel --pvalue=1e-2” and “--shiftsize=110 --mfold=10,10000 --slocal=2000 --llocal=20000" or ” --shiftsize=60 --mfold=4,10000" depending on the length distribution for DNA fragment sizes prior to sequencing. We also called peaks using Grizzly Peak fitting program {Capaldi:2008ff, Harrison:2011iy} with estimated DNA fragment length of 150 or 250.
We then intersected the two sets of peaks, and filtered out all peaks not supported by both methods. To account for low complexity peaks and possible PCR artifacts, we further removed peaks with negative correlation (<-0.1) among the Forward and Reverse reads, peaks where 60% of the reads mapped to less than 1% of the positions, and peaks whose height was less than three times the height of Input reads in the same locus.
We took as an initial dataset the union of all bound regions in the different replicates
Genome_build: Apr. 2006 assembly (Flybase Release 5) of the D. melanogaster genome, the February 2006 assembly (Flybase release 2) of the D.pseudoobscura genome and the February 2006 assembly (Flybase release 1) of the D.virilis genome
Supplementary_files_format_and_content: bwt files were generated using bowtie. They indicate the coordinates of mapped reads.
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| Submission date |
Sep 11, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Mathilde PARIS |
| E-mail(s) |
thildeparis@gmail.com
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| Organization name |
CNRS
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| Department |
Institut de Genomique Fonctionnelle de Lyon
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| Street address |
32-34 avenue Tony Garnier
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| City |
Lyon |
| ZIP/Postal code |
69006 |
| Country |
France |
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| Platform ID |
GPL17715 |
| Series (2) |
| GSE50771 |
Extensive divergence of transcription factor binding in Drosophila embryos with highly conserved gene expression (part 2) |
| GSE50773 |
Extensive divergence of transcription factor binding in Drosophila embryos with highly conserved gene expression |
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| Relations |
| BioSample |
SAMN02353808 |
| SRA |
SRX348466 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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