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| Status |
Public on Dec 22, 2017 |
| Title |
PFL_E12_delattp-rel5d9lac_Cs65 |
| Sample type |
SRA |
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| Source name |
Proximal Forelimbs
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| Organism |
Mus musculus |
| Characteristics |
tissue: Proximal Forelimbs
genotype: delHoxd(attP-rel5)d9lac
strain: B6CBAF1
embryonic day: E12.5
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Micro-dissected proximal and distal segments of E12.5 forelimbs either from wild type or mutant embryos (delHoxD(10-12), delHoxd(9-12), delHoxd(8-13)d11lac, delHoxd(8-13)rXII, delHoxd(1-13)d11lac, delHoxd(1-13)d9lac,delHoxD(attP-Rel5)d9lac). 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 at least 10 distal or proximal limbs 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. The barcode is mentioned in the raw file name being: A14 (ATCG), A24 (ACTT), C14 (CAGA), C44 (CTGT), G14 (GACA), G24 (GGTA), T14 (TGAC), T24 (TAGC) and T34 (TTAG). Specific primer information for each viewpoint is displayed in the related publication (Rodríguez-Carballo et al, 2017). Undigested and self-ligated fragments were discarded according to the sequences provided as an associated file named "Rodriguez-Carballo_2017_4C-seq_filtered_out_sequences.txt"
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Proximal forelimb mouse E12.5_del(attp-rel5)d9lac_Cs65 viewpoint
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| Data processing |
Library strategy: 4C-Seq
De-multiplexing, mapping and 4C-analysis were performed through HTSstation (David et al, 2014 and http://htsstation.epfl.ch/). Reads were mapped to the mm10 (GRCm38) mouse genome assembly with bowtie2 version 2.2.1 (Langmead et al, 2012) with parameters “--end-to-end --sensitive -k 20”. The segtofrag files were obtained after mapping the 4C data on in silico generated mutant genomes.
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.
segtofrag files were obtain after mapping the 4C on the in-silico generated mutant genomes (added as complementary file in Dryad -datadryad.org- or Figshare -figshare.com-) and performing the 4C-analysis on HTSstation (David et al, 2014 and http://htsstation.epfl.ch). The resulting data was used to compute the cumulative sum over the specified region (Rodriguez-Carballo et al, 2017).
Genome_build: mm10 (for bedGraph of smoothed/normalized data) and mutant genomes (available at DOI: 10.6084/m9.figshare.5220637) for segToFrag bedGraph.
Supplementary_files_format_and_content: Smoothed/normalized data (bedGraph), one per viewpoint, per tissue. Segtofrag are raw coverage of each fragment once mapped on the mutant genome.
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| Submission date |
Jul 20, 2017 |
| Last update date |
May 15, 2019 |
| 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) |
| GSE101713 |
A TAD boundary at the HoxD locus segregates opposing limb regulatory landscapes and their target genes [4C-Seq] |
| GSE101717 |
A TAD boundary at the HoxD locus segregates opposing limb regulatory landscapes and their target genes |
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| Relations |
| BioSample |
SAMN07374617 |
| SRA |
SRX3024487 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2713702_PFL_E12_delattp-rel5d9_CS65VP.bedGraph.gz |
4.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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