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Sample GSM1505557

Query DataSets for GSM1505557

Status

Public on Oct 20, 2014

Title

WT rep1

Sample type

SRA

Source name

16-18 hr embryo

Organism

Drosophila melanogaster

Characteristics

developmental stage: Embryo
age: 16-18 hr
genotype: w1118 (wild-type white)

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.

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.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

library strategy: Hi-C

Data processing

Paired-end reads were mapped to the Drosophila melanogaster genome (assembly dm3) using the bowtie program with parameters (-t -a -m 1 --best). 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 x Flen(alen,blen) x 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.
Genome_build: dm3
Supplementary_files_format_and_content: 50_200_top5.domains: a list of the physical domains index - unique ID for domain chrom - chromosome start - start co-ordinate of domain end - end co-ordinate of domain cluster - a number from 1 to 4 to identify the epigenetic nature of the domain. 1 = null, 2 = active, 3 = PcG, 4 = HP1/centromeric
Supplementary_files_format_and_content: *.bins: the real genomic co-ordinates of the involved bins cbin - the identity of the bin chr - chromosome from.coord - start co-ordinate of bin to.coord - end co-ordinate of the bin count - the number of fends contained within the bin
Supplementary_files_format_and_content: *.n_contact: give the contact score maps cbin1 - the identity of the first bin cbin2 - the identity of the second bin expected_count - the number of counts expected for this pairwise bin combination, based on the technical normalisation observed_count - the number of tags counted from the Hi-C dataset for this pairwise bin combination

Submission date

Sep 16, 2014

Last update date

May 15, 2019

Contact name

Jia-Ming Chang

E-mail(s)

[email protected]

Organization name

National Chengchi University

Department

Department of Computer Science