Gene Expression
Nebulas
Gene Expression NebulasSummary: Non-coding ultraconserved regions showing hundreds of consecutive bases of perfect evolutionary sequence conservation across mammalian genomes have intrigued biologists in the decade since they were first described. While many of these sequences are known to represent distant-acting enhancers, initial deletion studies in mice showed that their loss had no obvious impact on viability or fertility. To explore the discrepancy between extraordinary evolutionary constraint and an apparent lack of phenotypes when deleted in vivo, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved brain enhancers near the essential neuronal transcription factor Arx. While the loss of any single or pair of ultraconserved enhancers resulted in viable and fertile mice, detailed phenotyping revealed neurological or growth abnormalities in nearly all cases, including substantial alterations of neuron populations and abnormalities of the dentate gyrus. Our results demonstrate the functional importance of ultraconserved enhancers and highlight that extreme sequence conservation may result from evolutionary selection against fitness deficits that appear subtle in a laboratory setting.
Overall Design: Cell suspensions were prepared from E12.5 mouse forebrains, and single-cell mRNAseq libraries were generated with Drop-Seq. Experiments were performed on transgenics embryos in which a validated forebrain enhancer (hs119, hs121, hs122, hs123, http://enhancer.lbl.gov/) controlled the expression of a reporter gene (either GFP or mCherry).
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| Extract Protocol: | Total RNA was extracted using the RNAqueous Total RNA Isolation Kit (Thermo Fisher Scientific), then it was incubated with microbeads (ChemGene Beads, Lot 011416B).; Single-cell suspensions were processed through Drop-Seq to generate single-cell cDNA libraries attached to microbeads (ChemGene Beads, Lot 011416B). Cell concentration was set to 50 cells/碌l. 2.5% 293T/17 human cells were added to each suspension.As previously described (PMID: 26000488). |
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| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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