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| Status |
Public on Nov 10, 2020 |
| Title |
E9_FLB_wt_CTCF-37154 |
| Sample type |
SRA |
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| Source name |
Whole forelimb bud
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| Organism |
Mus musculus |
| Characteristics |
tissue: Whole forelimb bud
genotype: wild-type
strain: B6CBAF1
embryonic day: E9
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Micro-dissected proximal segments of E12.5 and E9 forelimbs either from wild type or mutant embryos. Tissues were dissected, dissociated with collagenase (Sigma Aldrich/Fluka) and filtered through a 35 micron mesh to isolate single cells. Cells were fixed with 2% formaldehyde (in PBS/10%FBS) for 10 min at room temperature and the reaction was quenched on ice with glycine. Cells were further lysed with 10 mM Tris pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1x Protease inhibitor cocktail to isolate nuclei and stored at -80°C. Nuclei from pools of 10-12 pairs of proximal limbs or 90 to 150 were digested with NlaIII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) in diluted conditions to promote intramolecular ligation. Samples were digested again with DpnII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) in diluted conditions.
These templates were amplified using Expand long template (Roche) and inversed PCR primers flanked with adaptors allowing multiplexing. The illumina adaptors used were 5'-AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' for the inverse-forward primers located at the NlaII site and 5'-CAAGCAGAAGACGGCATACGA-3' for the inverse-reverse primers located at the DpnII site. Barcodes (4bp) were added between the illumina adaptor and the specific NlaIII primers when the same viewpoints were multiplexed in the same run. Specific primer information for each viewpoint is displayed in the related publication (Rodríguez-Carballo et al, 2020).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Forelimb bud mouse E9_wt_CTCF-37154 viewpoint
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| Data processing |
Library strategy: 4C-seq
Analyses were performed using the facilities of the Scientific IT and Application Support Center of EPFL. De-multiplexing, mapping and 4C-analysis were performed through on a local version of HTSstation (David et al, 2014 and http://htsstation.epfl.ch/). Reads were mapped to the inv(T-DOM) mm10-based genome or the wild-type mm10 (GRCm38) mouse genome assembly with bowtie2 version 2.2.1 (Langmead et al, 2012) with parameters “--end-to-end --sensitive -k 20”.
segtofrag files were obtain after mapping the 4C on mm10 (or mm10_invTDOM) and performing the 4C-analysis of the local version of HTSstation (David et al, 2014 and http://htsstation.epfl.ch).
4C figures were made using a running mean algorithm with a window size of eleven fragments. Normalisation is done by dividing the fragment scores by the mean of fragments scores falling into a region defined as +/-5Mb around the centre of the bait coordinates, such as to see only changes in contact distribution and to minimize PCR complexity bias in each sample.
The segToFrag were smoothed on 11 fragments after the removing 0, 1kb, 2kb, 3kb each side of the viewpoint. 4Cin (Irastorza-Azcarate et al., 2018) was applied 5 times for each of the 4 preprocessing steps. The 20 virtual Hi-C obtained were then averaged and analysed for clustering.
all scripts are available at https://github.com/lldelisle/scriptsForRodriguezCarballoEtAl2020
Genome_build: mm10 or mm10_invTDOM
Supplementary_files_format_and_content: Smoothed/normalized data (bedGraph), one per viewpoint, per tissue. Segtofrag are scores of each fragment once mapped on the wild-type or mutant genome. AllVhic.tar.gz contains all virtual Hi-C as text files as output from 4Cin (20 per tissue-stage). Cool files are matrices summarizing all vitual Hi-C: *__all.cool is the average of all models and *__cluster*.cool is the average of all models in the same cluster.
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| Submission date |
Jul 10, 2020 |
| Last update date |
Nov 11, 2020 |
| Contact name |
Eddie Rodríguez-Carballo |
| E-mail(s) |
edgardo.rodriguez@unige.ch
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| Organization name |
Université de Genève
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| Department |
Department of Genetics and Evolution
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| Street address |
4, Boulevard d'Yvoy
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| City |
Geneva |
| ZIP/Postal code |
1205 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE154186 |
Chromatin topology and the timing of enhancer function at the HoxD locus [4C-seq] |
| GSE154189 |
Chromatin topology and the timing of enhancer function at the HoxD locus |
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| Relations |
| BioSample |
SAMN15502740 |
| SRA |
SRX8706902 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4665688_E9_FLB_wt_CTCF-37154.bedGraph.gz |
638.2 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM4665688_segToFrag_E9_FLB_wt_CTCF-37154.bw |
150.4 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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