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| Status |
Public on Jun 08, 2016 |
| Title |
Dissecting the direct reprogramming path of fibroblasts into neurons by single cell RNA-sequencing |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediates through which individual cells progress are largely undefined. Here we used single cell RNA-seq at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity rather than time we reconstructed a continuous reprogramming path. We find that overexpression of a single factor (Ascl1) results in a well-defined initialization causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. However, overexpression of Ascl1 alone leads to abundant alternative fates that are suppressed by the combination of additional factors (Myt1l, Pou3f2). We find transgene silencing and emergence of alternative fates are the major efficiency limits of direct reprogramming. These data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.
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| Overall design |
405 single-cell transcriptomes from 5 time points (0 days, 2 days, 5 days, 20 days and 22 days upon Ascl1 induction) during reprogramming of MEFs into iN cells were analyzed in total; All single cells contain 92 external RNA spike-ins; The following single cell transcriptomes were sequenced: 73 single MEF cells (day 0, iN2), 81 single cells at day 2 (iN1), 55 single cells at day 5 (iN3), 33 single cells at day 20 (iN6) and 73 single cells at day 22 (iN7,iN8) upon Ascl1 induction from virally transfected MEFs. Additionally, we generated the transcriptome of 47 single cells that were induced with Ascl1 for 2 days from a clonal MEF line with a uniform Ascl1 transgene copy number (iN5). Finally, we sequenced the transcriptome of 43 single cells at day 22 (iN4) upon induction of Ascl1, Brn2 and Myt1l. All single cell samples were processed on the microfluidic Fluidigm C1 platform.
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| Contributor(s) |
Treutlein B, Quake SR |
| Citation(s) |
27281220 |
| Submission date |
Mar 26, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Barbara Treutlein |
| E-mail(s) |
baerbel.treutlein@gmail.com
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| Organization name |
Stanford University
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| Department |
Bioengineering
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| Lab |
Quake
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| Street address |
318 Campus Drive
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| City |
Stanford CA |
| State/province |
CA |
| ZIP/Postal code |
94305-5432 |
| Country |
USA |
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| Platforms (2) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (405)
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| Relations |
| BioProject |
PRJNA279486 |
| SRA |
SRP056585 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE67310_iN_data_log2FPKM_annotated.txt.gz |
17.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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