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| Status |
Public on Dec 06, 2016 |
| Title |
CC-HL-E105-Wt-Mm-Rep2 |
| Sample type |
SRA |
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| Source name |
Embryonic Limb Bud
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Hindlimb
developmental stage: E10.5
genotype: wild type
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| Growth protocol |
Limb buds and midbrain were micro-dissected from mouse embryos
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The 3C library was prepared according to (Hagege et al. 2007), i.e. crosslinking, cell lysis, DpnII digestion, ligation and de-crosslinking. Re-ligated products were then sheared using a covaris sonicator (Duty cycle: 10%, Intensity:5, Cycles per Burst: 200, Time: 6 cycles of 60 sec each, Set Mode: Frequency sweeping, Temperature: 4° to 7°C).
Adaptors were added to the sheared DNA and amplified. The adapted-ligated library was then hybridized to the custom-designed sure-select beads indexed for sequencing using an indexing PCR following Agilent instructions.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Captured genomic DNA from 3C library
Processed data file for smoothed signal, peaks, and enhancer data are located on GSM2251420
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| Data processing |
Library strategy: Capture-C
Frag profiles: Paired-end sequencing data from Capture-C experiments was mapped and cleaned from invalid di-tags and duplicates using the HiCUP pipeline v0.5.8 (Wingett et al. 2015) and Bowtie 2.2.6 (Langmead and Salzberg 2012). All sequencing data was mapped to mouse reference genome mm9. Viewpoint-specific contact profiles were generated from BAM files with custom Java code using htsjdk 1.139 (https://samtools.github.io/htsjdk/) for the processing of BAM files. A di-tag was considered only if one mate was located in an enriched viewpoint region while the other mapped to the non-enriched part of the genome. Contact profiles were generated for each viewpoint by counting the number reads per restriction fragment. Count data was binned into a regular grid of 1 kb intervals centered around the middle of the corresponding viewpoint region. The count value of each restriction fragment was assigned to the bin, which contains the fragment center.
Smoothed profiles: count data per fragments was combined from both replicates, binned, smoothed with a running window of 3 and 5 bins. To make contact profiles with different read densities comparable, each profile was scaled by the total contact count on the chromosome of the viewpoint (Sum of cis contacts/1000).
Peak calling: Scaled contact profiles were pooled across all samples and viewpoints to generate a simple, empirical background model, which incorporates the distance dependent decay of the contact frequency. The positioning of the grid around viewpoint center allows an overlay of all profiles by aligning their centers. Now, each 1 kb bin can be described by its offset relative to the viewpoint center and has the contact information from 6244 profiles (7 tissues/stages × 446 viewpoints × 2 replicates). The 99th and 95th percentile of the empirical distribution were used to derive an individual threshold for each bin. A spline function was used to smooth the resulting threshold line. To avoid the calling of spurious interactions due to a lower profile density at locations distant from the viewpoint, the threshold was set constant for all regions more than 0.5 Mb away from the viewpoint. A bin was selected as candidate region, when the contact value exceeds the threshold in both replicates. Within the candidate region the maximum value from the merged profile was used to determine the peak center. A region +-10 kb around the selected peak was excluded from the candidate region to suppress peaks nearby. The selection of maxima and exclusion of sub-peaks was repeated until no further candidates were left.
Enhancer calling: Putative enhancers were called based on the combination of three thresholds: the 95th percentile of the empirical background for the interaction strength, the 99th percentile for H3K27Ac and 95th percentile for H3K4me1. Percentiles of the histone modifications were computed based on genome wide distribution of mapped reads within 5 kb windows.
Genome_build: mm9
Supplementary_files_format_and_content: Bed and bedgraph files were generated in each sample for 446 different viewpoints and packed for each analysis into one archive. Smoothed profiles, peak files and enhancer files contain data summarized across the two replicates. The files are located on the 'Rep1' sample.
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| Submission date |
Jul 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Guillaume Andrey |
| E-mail(s) |
guillaume.andrey@unige.ch
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| Phone |
+41223795703
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| Organization name |
University of Geneva
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| Department |
Department of Genetic Medicine and Development
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| Street address |
Rue Michel-Servet 1
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| City |
Geneva |
| ZIP/Postal code |
1211 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE84792 |
Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding [Capture-C] |
| GSE84795 |
Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding |
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| Relations |
| BioSample |
SAMN05439068 |
| SRA |
SRX1975543 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2251421_CC-HL-E105-Wt-Mm_Frag-Rep2.tar.gz |
16.4 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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