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| Status |
Public on Jul 02, 2016 |
| Title |
Nppa viewpoint in the E12.5 ventricles |
| Sample type |
SRA |
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| Source name |
embryonic heart ventricles
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| Organism |
Mus musculus |
| Characteristics |
tissue: heart ventricles
strain: FVB/N
Stage: E12.5
bait: chr4_Nppa_147374910
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| Treatment protocol |
cells are cross linked using 2% formaldehyde for 10minutes at room temperature in 10%FCS/PBS
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| Growth protocol |
control and TAC-operated mice were used, where TAC (transverse aortic constriction) induces cardiac hypertrophy
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
4C templates was prepared as described previously (Simonis et al., 2009). Adult mouse hearts and liver were dissociated and homogenized in ice-cold PBS with 10%FCS to obtain single cell suspension. Chromatin was cross-linked with 2% formaldehyde in 40ml PBS with 10% FCS for 10 min at room temperature, nuclei were isolated in 25ml cold lysis buffer for 1h, and cross-linked DNA was digested with DpnII. Digestion was followed by proximity ligation, removal of cross-links, a secondary restriction digestion with Csp6I and a second proximity ligation.
200 ng of the resulting 4C template was used for the subsequent PCR reaction using Expand Long Template Polymerase (Roche). The PCR products were purified using the High Pure PCR Product Purification Kit (Roche) and the QIAquick PCR purification kit (Qiagen).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Library strategy: 4C-seq
4C templates were mixed and sequenced simultaneously in 1 Illumina HiSeq 2000 lane. The sequence tags generated by the procedure were prefixed by the 4C reading primer, which includes the DpnII restriction site sequence. The 4C reading primer sequences were separated from multiplexed 4C-seq libraries, and the suffixes were extracted for further processing.
Mapping and filtering of the sequence reads was done as previously described (Werken et al., 2012, Nat Methods). The algorithm constructs a background model for remote intra- and interchromosomal contacts to correct for systematic biases that can occur during the 4C-seq experimental protocol. The algorithm is designed to use controls for sequencing errors and nonunique sequences while considering the high coverage (100×–100,000×) of fragment ends that are proximal to the viewpoint fragment.
To normalize the interactions in close proximity to the viewpoint, the algorithm was used to calculate the median of normalized coverage for running windows of size 4 kb and sliding windows of 2–50 kb of linearly increasing size. All median values represent enrichment relative to the maximum attainable 4-kb median value, whereas sliding windows represent enrichment relative to the maximum attainable 12-kb median value. The 20th and 80th percentiles were also computed and depicted as the gray area around the 4-kb running windows.
The 4C-seq contact profile comparison plots were generated combining the mapping (Werken et al., 2012, Methods Enzymol) and normalization (Werken et al., 2012, Nat Methods) strategies. Within samples the distribution of the blind fragment-end reads and the distribution of the non-blind fragment-end reads were quantile normalized after linear interpolation of the center of the fragment-end using R's limma package (Smyth, 2005). The reads between samples were normalized based on library size within the locus. Subsequently, a running trimmed (10%) mean was calculated on 21 fragment-ends to smoothen the 4C-seq data. The R statistical package version 3.1.0 was used for the statistical calculations and for generating the 4C-Seq plots (R Core Team, 2013).
Genome_build: mm9
Supplementary_files_format_and_content: Wiggle tracks contain uniquely mapped reads at 4C fragment ends on the cis chromosome, i.e. the chromosome on which the bait/viewpoint fragment is located (chr4). Scores represent the number of reads mapping to a DpnII restriction fragment end.
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| Submission date |
Jul 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Vincent M. Christoffels |
| E-mail(s) |
v.m.christoffels@amsterdamumc.nl
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| Phone |
0205667821
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| Organization name |
Amsterdam UMC
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| Department |
Medical Biology
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| Lab |
Vincent Christoffels
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| Street address |
Meibergdreef 15 K2-159
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| City |
Amsterdam |
| State/province |
N/A (NA) |
| ZIP/Postal code |
1105 AZ |
| Country |
Netherlands |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE81057 |
Identification of a regulatory domain controlling the Nppa-Nppb gene cluster during heart development and stress |
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| Relations |
| BioSample |
SAMN05346353 |
| SRA |
SRX1892884 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2224631_FVB_heart_E12.5_Nppa_chr4_win_21_cis.wig.gz |
1.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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