| Series | GSE69790 |
| Title | Single cell transcriptomics analysis of induced pluripotent stem cell-derived cortical neurons reveals frequent dual layer identity |
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| Year | 2016 |
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| Country | United Kingdom |
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| Article | Cader MZ,Ponting CP,Akerman C,Newey S,Cowley S,Bowden R,Nakanishi M,Sopp P,Argoud K,Giustacchini A,Vowles J,Whiteley E,Lalic T,Chintawar S,Handel AE.Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics.Human molecular genetics.2016 Mar 1 |
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| PMID | 26740550 |
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| Bio Project | BioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA286775 |
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| Sra | SRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP059379 |
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| Overall Desgin | Single cell RNA-seq of 16 iPSC-derived cortical neurons. |
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| Summary | 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. |
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| Experimental Protocol | Cells were dissociated as for single cell RT-qPCR at day 72 of neuronal differentiation. 300,000 DAPI negative cells were sorted into 200ïL 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.; We continued with lysis, reverse transcription, amplification and library prep in accordance with the Fluidigm Smarter-seq protocol.
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| Data processing | Trimmomatic with the arguments “LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:50â€; Tophat2; Rsubread; DESeq adjustment for library size and ERCC counts from 92 ERCC (External RNA Control Consortium) controls; Genome_build: hg19 plus ERCC 92; Supplementary_files_format_and_content: adjusted counts (tsv)
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| Platform | GPL11154 |
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| Public On | Public on Jan 08 2016 |
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