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SRX9418426: GSM4876557: E14_H3K27me3_2; Mus musculus; OTHER
1 ILLUMINA (Illumina NovaSeq 6000) run: 185.1M spots, 28G bases, 10.4Gb downloads

Submitted by: NCBI (GEO)
Study: The chromatin and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo [HiChIP]
show Abstracthide Abstract
Transcriptional and phenotypic robustness during development is believed to require complex regulatory landscapes whereby multiple enhancers redundantly control the expression of major cell identity genes. In contrast, we previously described a limited and genetically distinct set of distal regulatory elements, known as poised enhancers (PEs), that control the induction of genes involved in early brain development in a hierarchical and non-redundant manner. Before becoming activated in neural progenitors, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally identified and subsequently characterized using embryonic stem cells (ESC) as in an in vitro differentiation system, it is currently unknown whether PEs are functionally conserved in vivo. Here, we generate and mine various types of genomic data to conclusively show that the epigenetic and 3D structural features of PEs are conserved among pluripotent cells both in vitro and in vivo. Furthermore, by genetically disrupting evolutionary conserved PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential and non-redundant roles during the induction of major anterior neural genes in vivo. Overall design: Chromosome confirmation capture for various histone modifications were investigated in mESC (undifferentiated and differentiated), as well as early murine and avian embryonic stages using HiChIP.
Sample: E14_H3K27me3_2
SAMN16624749 • SRS7633771 • All experiments • All runs
Organism: Mus musculus
Library:
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Fertilized chicken eggs (white leghorn; Gallus gallus domesticus) were obtained from a local breeder (LSL Rhein-Main) and incubated at 37°C and 80% humidity in a normal poultry egg incubator (Typenreihe Thermo-de-Lux). Following microsurgical procedures, the eggs were re-incubated until the embryos reached the desired developmental stages. The developmental progress was determined according to the staging system of HH (Hamburger and Hamilton, 1992). Mice were housed and bred under standard conditions in the CECAD ivRF. The breedings described were approved by the Landesamt für Natur, Umwelt, und Verbraucherschutz Nordrhein-Westfalen (LANUV), Germany (animal application 84-02.04.2015.A405). Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibodies againts the specific proteins of interest in each case. HiChIP was performed as described by Mumbach et al. 2016 Nat Methods with some modifications. We generally replaced the ChIP protocol with the one described above, we cut and ligated overnight (NEB T4 Ligase, instead of Invitrogen T4) and DNA was extracted with phenol-chlorophorm not a kit. For low cell numbers, we increased the centrifugation time to 30 minutes and 15 minutes after lysis, as well as 30 minutes after ligation to see a pellet more accurately. Generally 12 cycles were used for Tn5 Nextera PCR amplification (Illumina Nextera DNA UD Indexes Kit). We aimed for 100M read pairs for each run on a NovaSeq 6000 sequencer (Illumina).
Experiment attributes:
GEO Accession: GSM4876557
Links:
Runs: 1 run, 185.1M spots, 28G bases, 10.4Gb
Run# of Spots# of BasesSizePublished
SRR12965873185,136,95328G10.4Gb2021-01-01

ID:
12299624

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