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| Status |
Public on Jan 20, 2012 |
| Title |
Deep-sequenced simplified Hi-C |
| Sample type |
SRA |
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| Source name |
16-18 hr embryos
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: Embryo
age: 16-18 hr
genotype: Oregon-R w1118
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| Growth protocol |
Oregon-R w1118 flies were raised in standard cornmeal yeast extract media at 25°C. Egg laying was performed on plates with standard vinegar medium and yeast. Embryos were collected in 0.03% Triton-X100, 0.4% NaCl 16-18 hr after egg laying.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
~3000-4000 embryos were dechorionated for 5 min in fresh bleach, before douncing in 5 ml fix buffer (2% formaldehyde, 15 mM HEPES pH 7.6, 60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 0.1% Triton-X100, 0.5 mM DTT, protease inhibitor cocktail (Roche)) and fixed for a total of 10 min at 25°C, 750 rpm on a shaker. After quenching with 5 ml 2 M glycine, nuclei were collected by centrifugation for 5 min at 4500 g, 4°C, then washed once with 5 ml fix buffer (without formaldehyde) and once with 1.25 x NEB3 buffer (New England Biolabs), with centrifugation for 5 min at 4500 g, 4°C each time. Nuclei were resuspended in 300 µl 1.25 x Dpn II buffer (New England Biolabs) plus 0.3% SDS and incubated for 1 hr at 37°C, 1000 rpm on a shaker. Triton-X100 was added to a final concentration of 2% and the nuclei were incubated for a further 1 hr at 37°C, 1000 rpm, before overnight digestion with 1500 U Dpn II (New England Biolabs) at 37°C, 1000 rpm. The restriction enzyme was inactivated by incubation for 20 min at 65°C, 1000 rpm with SDS at a final concentration of 1.3%, before dilution of the chromatin in 10 ml 1x T4 DNA ligase buffer (New England Biolabs) plus 1% Triton-X100 and incubation for 1 hr at 37°C, 750 rpm. The chromatin was ligated for 4 hr at 25°C, 750 rpm with 40,000 U T4 DNA ligase (New England Biolabs) then crosslinks were reversed overnight at 65°C, 750 rpm in the presence of 150 µg/ml proteinase K. The 3C DNA was then purified by 1 hr treatment with 40 µg/ml RNase A at 37°C, 750 rpm, phenol extraction, phenol/chloroform extraction and ethanol precipitation. The DNA was quantified with the Qubit dsDNA assay (Invitrogen). Aliquots of 5 µg 3C DNA were sonicated in a Bioruptor (Diagenode) in 50 µl volumes in sonication buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS) to obtain a smear between 500-1500 bp. The sonicated 3C DNA was then purified by phenol/chloroform extraction and ethanol precipitation and quantified with the Qubit dsDNA assay (Invitrogen). Libraries for paired-end sequencing were made from 500 ng aliquots of sonicated 3C DNA using Illumina reagents and protocols, with size selection for products of ~800 bp for the deep-sequenced sample, or 300-800 bp for the pilot sample.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
deep10k_normalized.txt; genome build: dm3
deep20k_normalized.txt; genome build: dm3
deep40k_normalized.txt; genome build: dm3
deep80k_normalized.txt; genome build: dm3
deep160k_normalized.txt; genome build: dm3
Paired-end reads were mapped to the Drosophila melanogaster genome (assembly dm3) using the maq program with standard parameters (quality>30). Unmapped or non-unique mapped reads were discarded. We then divided read pairs into four groups: SS (reads mapping to the same restriction fragment, or to two adjacent restriction fragments); S2 (reads in which both ends mapped precisely on the DpnII site); S1 (reads in which one of the ends mapped precisely on the DpnII site); S0 (reads in which both ends did not map precisely to a DpnII site). SS, S2 and S1 pairs were discarded from subsequent analysis. Mapped reads were then associated with the first restriction fragment end encountered when moving from the mapped position in the appropriate strand. All subsequent analysis was done in the space of fragment end pairs. The Drosophila genome contains 464,246 restriction fragments. After discarding low mappability fragments 301,687 fragments are left (and each defines two fragment ends). A model accounting for systematic biases in contact probability was constructed as previously (Yaffe and Tanay; Nature Genetics, 43, p.1059-65). Briefly, we used the local G+C content of up to 200 bp from the restriction site (agc, bgc), and the DpnII restriction fragment lengths (alen, blen), to correct for systematic biases in Hi-C coverage by modeling: P(Xa,b) = Pprior . Flen(alen,blen) . Fgc(agc,bgc). We estimated maximum likelihood model parameters (the matrices Flen and Fgc) through application of an iterative algorithm as described, using 20 bins for G+C content and 20 bins for fragment length.
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| Submission date |
Dec 14, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Tom Sexton |
| E-mail(s) |
thomas.sexton@igh.cnrs.fr
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| Organization name |
Institute of Human Genetics, CNRS UPR 1142
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| Street address |
141, rue de la Cardonille
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| City |
Montpellier |
| ZIP/Postal code |
34396 |
| Country |
France |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE34453 |
Three-dimensional folding and functional organization principles of the Drosophila genome |
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| Relations |
| SRA |
SRX111555 |
| BioSample |
SAMN00765544 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM849422_deep10k_normalized.txt.gz |
11.9 Mb |
(ftp)(http) |
TXT |
| GSM849422_deep160k_normalized.txt.gz |
3.5 Mb |
(ftp)(http) |
TXT |
| GSM849422_deep20k_normalized.txt.gz |
7.2 Mb |
(ftp)(http) |
TXT |
| GSM849422_deep40k_normalized.txt.gz |
7.6 Mb |
(ftp)(http) |
TXT |
| GSM849422_deep80k_normalized.txt.gz |
14.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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