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SRX6958289: GSM4110057: GT_E13_5_WT_rep2_Hoxd13VP; Mus musculus; OTHER
1 ILLUMINA (Illumina HiSeq 2500) run: 4.2M spots, 124.9M bases, 73.1Mb downloads

Submitted by: NCBI (GEO)
Study: A complex regulatory landscape involved in the development of external genitals [4C-seq]
show Abstracthide Abstract
In vertebrates, developmental genes are often controlled by large regulatory landscapes matching the dimensions of topologically associating domains (TADs). In various ontogenic contexts, the associated constitutive chromatin backbone is modified by fine-tuned specific variations in enhancer-enhancer and enhancer-promoter interaction profiles. In this work, we use a TAD flanking the HoxD gene cluster as a paradigm to address the question of how these complex regulatory architectures are formed and how they are de-constructed once their function has been achieved. We suggest that this TAD can be considered as a coherent functional unit in itself, with several regulatory sequences acting together to elicit a transcriptional response. With one notable exception, the deletion of each of these sequences in isolation did not produce any substantial modification in the global transcriptional outcome of the system, a result at odds with a conventional view of long-range enhancer function. Likewise, both the deletion and inversion of a CTCF site located in a region rich in such sequences did not affect transcription of the target gene. In the latter case, however, slight modifications were observed in interaction profiles in vivo in agreement with the loop extrusion model, despite no apparent functional consequences. We discuss these unexpected results by considering both conventional explanations and an alternative possibility whereby a rather unspecific accumulation of particular factors within the TAD backbone may have a global impact upon transcription. Overall design: 4C-seq analysis of 3D chromatin interactions in different wild type and mutant mouse embryonic tissues
Sample: GT_E13_5_WT_rep2_Hoxd13VP
SAMN12982938 • SRS5485113 • All experiments • All runs
Organism: Mus musculus
Library:
Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: Circular chromosome conformation capture (4C-seq) was performed as described in (Noordermeer et al., 2011). Briefly, tissues (20-40 GT or 40 cloaca) were isolated in PBS supplemented with 10% Fetal Calf Serum and dissociated to single cell by collagenase treatment. Samples were fixed in 2% formaldehyde, lysed, and stored at −80°C. Samples were primarily digested with NlaIII (NEB, R0125L) followed by ligation under diluted conditions. After decrosslinking and DNA purification DpnII (NEB, R0543M) was used for the second restriction. All ligation steps were performed using highly concentrated T4 DNA ligase (Promega, M1794). For each viewpoint approximately 1μg of DNA was amplified by using 12 individual PCR reactions. Libraries were constructed with inverse primers for different viewpoints (see Table 5) containing Illumina Solexa adapter sequences and sequenced on an Illumina HiSeq 2500 sequencer, as single-end reads (read length 100 base pairs or 80 base pairs). In some samples 4-bp barcodes were added between the adapter and each specific viewpoint to allow sample multiplexing.
Experiment attributes:
GEO Accession: GSM4110057
Links:
Runs: 1 run, 4.2M spots, 124.9M bases, 73.1Mb
Run# of Spots# of BasesSizePublished
SRR102391094,163,199124.9M73.1Mb2020-04-25

ID:
9155283

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