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Sample GSM3619420

Query DataSets for GSM3619420

Status

Public on Jan 12, 2020

Title

HiC_ED_rep3_a

Sample type

SRA

Source name

HiC_ED

Organism

Drosophila melanogaster

Characteristics

strain: W1118 Oregon-R
develpmental stage: L3 wandering larvae
tissue: Eye Disc

Extracted molecule

genomic DNA

Extraction protocol

Hi-C experiments from EDs and 16-18h embryos were conducted as previously described in Ogiyama, et al. 2018. For larval Hi-C triplicates, ~200 EDs were quickly dissected (<1h) from wandering larvae at R.T in Schneider’s insect medium before being directly processed. Briefly, formaldehyde was added to reach a 2% final concentration and the samples were homogenized for 3 minutes using a tight tenbrock at R.T and transferred on a rotating wheel (total time for fixation is 10min). Then, cell membrane was lysed on ice and nuclei were subjected to DpnII treatment O/N at 37°C. The next day, restriction sites were end-repaired and biotinylated using Klenow (NEB, M0210) and biotin-14-dATP (Life Tech., 19524-016) before being re-ligated in situ using T4 DNA ligase (NEB, M0202). Then, proteins were degraded with proteinase K before O/N de-crosslinking at 68°C and subsequent DNA purification using Phenol/chloroform and ethanol precipitation. Purified DNA was sheared with a LE220 Covaris sonicator (volume: 130µl, fill lvl: 10, duty cycle: 15, PIP: 500, cycles/burst: 200, time: 58s). Finally, sonication efficiency was checked on an agarose gel.
DNA fragments were size-selected using AMPure XP beads (300-500bp), and ligation fragments containing biotin were immobilized on MyOne Streptavidin T1 beads (Life Technologies, 65602). Biotin was removed from unligated ends, and pulled-down DNA fragments were end-repaired and a-tailed before NEXTflex (Bioo Scientific, 514101) adaptors ligation. Finally, libraries were PCR amplified (10-12 cycles) using NEBNext® Ultra™ II Q5® Master Mix (New England Biolabs, M0544L) and DNA was then double-size selected using AMPure XP beads (Agencourt, Cat.N: A63881) in order to isolate fragments between 300 and 800bp.

Library strategy

Hi-C

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

processed data file: HiC_ED_scores.tar.gz
run1

Data processing

Raw Hi-C sequencing data were processed using the “scHiC2” pipeline (Nagano et al., 2017). Construction of expected models, Hi-C contact scoring and Hi-C aggregate plots was performed using the “shaman” R package (https://bitbucket.org/tanaylab/shaman, Cohen et al., bioRxiv, 2017). Hi-C map visualization, segmented interaction frequencies and downstream analyses were conducted using in-house R scripts using the “misha” package (https://github.com/msauria/misha-package).
Raw sequencing data were processed using the HiC-Pro pipeline (version 2.11.1, REF: 26619908) with default parameters and the dm6 reference genome. Resulting raw data were normalized at 2, 5 and 10kb resolutions using the ice_norm flag. In parallel, the hicpro2juicebox utility was used to convert the allValidPairs output of the pipeline into the Juicebox .hic format at fragment resolution.
Supplementary_files_format_and_content:
bw files correpond to the merged (2 replicates) RPKM-normalized bigwig binary files (10bp bins), which can be visualized using the Integrative Genomics Viewer (IGV) software. NarrowPeak files correpond to the peaks that were called using the MACS2 software on separated replicates and on the merged replicates (10 columns: chrom, chromStart, chromEnd, name, score, strand, signalValue, pValue, qValue, peak). Summit file contains the summits of the peaks called from the merged replicates.
Compressed HiC tracks; Binary files of intrachromosomal pairwise interaction probabilities (score) computed using the "shaman" package in R.
Ouput file of differential expression analysis using the DESeq2 R package. 8 Columns: FBgn (FlyBase Gene ID); Symbol; baseMean; log2FoldChange; lfcSE; stat; pvalue; padj.
The .hic binary files can be visualized using Juicer (https://github.com/aidenlab/juicer). The bins_ids.bed, iced.matrix.biases and iced.matrix files contain the genomic coordinates of the bins, the bin bias used to normalize the observed data and the normalized HiC matrices in plain text format, respectively.
Genome_build: dm6

Submission date

Feb 24, 2019

Last update date

Jan 12, 2020

Contact name

Vincent Loubiere

E-mail(s)

vincent.loubiere@imp.ac.at

Phone

+33663182164

Organization name

UMR9002; CNRS-UM