Gene Expression
Nebulas
Gene Expression NebulasSummary: Long non-coding RNAs (lncRNAs) are a diverse category of transcripts with poor conservation and have expanded greatly in primates; particularly in their brain. We identified a lncRNA; which has acquired 16 microRNA response elements (MREs) for miR-143-3p in the Catarrhini branch of primates. This lncRNA termed LncND (neuro-development) gets expressed in neural progenitor cells and then declines in mature neurons. Binding and release of miR-143-3p; by LncND; can control the expression of Notch. Its expression is highest in radial glia cells in the ventricular and outer subventricular zones of human fetal brain. Down-regulation of LncND in neuroblastoma cells reduced cell proliferation and induced neuronal differentiation; an effect phenocopied by miR-143-3p over-expression and supported by RNA-seq analysis. These findings support a role for LncND in miRNA-mediated regulation of Notch signaling in the expansion of the neural progenitor pool of primates and hence contributing to the rapid growth of the cerebral cortex.
Overall Design: Cerebral organoids were generated as in Lancaster et al. (Lancaster and Knoblich; 2014). Organoids were dissociated into single cells and captured on C1 Single-Cell Auto Prep Integrated Fluidic Circuit (IFC) (Fluidigm). The RNA extraction and amplification was performed on the chip as described by the manufacturer. We captured 68 single-cells on a C1 Single-Cell Auto Prep System (Fluidigm) and sequenced the RNA on a NextSeq500 System (Illumina) (Pollen et al.; 2014). Out of 68 cells; we obtained 60 high quality cells.
| Strategy: |
|
| Species: |
|
| Tissue: |
|
| Healthy Condition: |
|
| Growth Protocol: | Cerebral organoids were generated as in Lancaster?et al. (Lancaster and Knoblich, 2014).?" |
| Treatment Protocol: | Briefly, human iPS cells were isolated in single-cell solution and plated in a 96-well ultra-low attachment plate in hESC media with ROCK-inhibitor and low bFGF for 5 to 6 days to form embryoid bodies (EBs). EBs were then transferred to 24-well ultra-low attachment plates in neural induction media containing N2 supplement for 4 to 5 days to form neuroepithelium, then embedded in matrigel droplets and cultured in 60 mm dishes in organoid differentiation media containing N2 and B27 supplements. Vitamin A was added after 4 days in organoid differentiation media, and organoids were incubated until indicated time points on an orbital shaker. |
| Extract Protocol: | Cerebral organoids were dissociated into single cells and captured on C1 Single-Cell Auto Prep Integrated Fluidic Circuit (IFC) (Fluidigm). The RNA extraction and amplification was performed on the chip as described by the manufacturer. |
| Library Construction Protocol: | cDNA was processed to prepare libraries using Nextera XT DNA Sample Preparation kit (Illumina) to sequence them on NextSeq500 platform (Illumina). |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Unspecific |