Gene Expression
Nebulas
Gene Expression NebulasSummary: Induced pluripotent stem cell (iPSC)-derived cortical neurons present a powerful new model of neurological disease. Previous work has established that differentiation protocols produce cortical neurons but little has been done to characterise these at cellular resolution. In particular; it is unclear to what extent in vitro two-dimensional; relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single cell multiplex RT-qPCR was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Unexpectedly; 22.7% of neurons analysed frequently co-expressed canonical fetal deep and upper cortical layer markers; and this co-expression was also present at the level of translated protein. By comparing our results to available single cell RNA-seq data from human fetal and adult brain; we observed that this co-expression of layer markers was also seen in primary tissue. These results suggest that establishing neuronal layer identity in iPSC-derived or primary cortical neurons using canonical marker genes transcripts is unlikely to be informative.
Overall Design: This dataset was used for normalization purposes for GSE67835.
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| Growth Protocol: | iPSCs were differentiated into cortical neurons using the Livesey protocol with some modifications (Shi et al. Nat. Protocol. 2012;7(10):1836–1846). In brief, iPSCs were cultured in monolayer on matrigel. Cells were induced using dual SMAD inhibition (1?M dorsomorphin and 10?M SB431542) in neural maintenance media (DMEM/F-12, neurobasal, N-2, B-27, 5?g/ml insulin, 1mM L-glutamine, 100?M non-essential amino acids, 100?M 2-mercaptoethanol, 50 units/ml penicillin and 50mg/ml streptomycin). After the formation of a neuroepithelial sheet, this was passaged into wells plated with L-ornithine and laminin. The subsequent differentiating cells were passaged after different durations in culture to expand cortical neural progenitor stocks. RT-qPCR and immunofluorescence microscopy confirmed the adoption of cortical identity. Cells were treated with 4?M cytosine arabinoside for 72 hours prior to single cell analysis. |
| Treatment Protocol: | - |
| Extract Protocol: | Cells were dissociated as for single cell RT-qPCR at day 72 of neuronal differentiation. 300,000 DAPI negative cells were sorted into 200 L of neural maintenance media. These were loaded onto a small C1 chip according to the Smarter-seq protocol detailed by Fluidigm. Cells were co-stained with Hoechst and propidium iodide. Capture chambers were imaged on an Opera Imaging System. |
| Library Construction Protocol: | We continued with lysis, reverse transcription, amplification and library prep in accordance with the Fluidigm Smarter-seq protocol. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Unspecific |