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| Status |
Public on Dec 07, 2016 |
| Title |
A multi-step transcriptional and chromatin cascade underlies motor neuron programming (ATAC-Seq) |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
Direct programming via the overexpression of transcription factors (TFs) aims to control cell fate at a precision that will be instrumental for clinical applications. However, direct programming of terminal fates remains an obscure process. Taking advantage of the rapid and uniquely efficient programming of spinal motor neurons by overexpression of Ngn2, Isl1 and Lhx3, we have characterized gene expression, chromatin and transcription factor binding time-course dynamics during complete motor neuron programming. Our studies point to a surprisingly dynamic programming process. Promoter chromatin and expression analysis reveals at least three distinct phases of gene activation, while programming factor binding shifts from one set of targets to another, controlling regulatory region activity and gene expression. Furthermore, our evidence suggest that the enhancers and genes activated in the final stage of motor neuron processing are dependent on the combined activities of Isl1 and Lhx3 factors with Ebf and Onecut TFs that are themselves activated midway through the programming process. Our results suggest an unexpected multi-stage model of motor neuron programming in which the programming TFs require activation of a set of intermediate regulators before they complete the programming process.
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| Overall design |
50,000 cells were harvested and washed in ice-cold PBS buffer. After centrifugation the supernatant was aspirated and the cell pellet was resuspended in a master mix of 22.5 µl RNase-free water, 25 µl TD buffer and 2.5 µl TDE1 (transposase enzyme, both Illumina Nextera DNA Library Prep kit), followed by incubation for 60 minutes at 37oC. The reaction was then cleaned using the DNA Clean and Concentrator-5 kit (Zymo Research). The optimal number of PCR cycles was determined to be Ct+4. qPCR reactions were performed using 10% of a master mix of sample, forward and reverse (barcode) primers, 1X SYBR Green I (Biozym) and 1X NEBNext PCR MasterMix (New England Biolabs). Following PCR amplification, the library was cleaned using the DNA Clean and Concentrator-5 kit and eluted in 15 µl 10 mM Tris-Cl pH 8.0. Sample was quantified using Qubit (Life Technologies) measurement and the fragment length distribution was determined using the Bioanalyzer DNA High Sensitivity assay (Agilent). Sequencing was performed on an Illumina NextSeq 500 using V2 chemistry for 150 cycles (paired-end 75nt).
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| Contributor(s) |
Velasco S, Ibrahim MM, Kakumanu A, Garipler G, Al-Sayegh MA, Hirsekorn A, Satija R, Ohler U, Mahony S, Mazzoni EO |
| Citation(s) |
27939218 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R01 HD079682 |
Molecular mechanisms of direct neuronal programming |
NEW YORK UNIVERSITY |
Mazzoni |
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| Submission date |
Apr 21, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Shaun Mahony |
| E-mail(s) |
mahony@psu.edu
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| Phone |
814-865-3008
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| Organization name |
Penn State University
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| Department |
Biochemistry & Molecular Biology
|
| Lab |
Shaun Mahony
|
| Street address |
404 South Frear Bldg
|
| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (4)
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| This SubSeries is part of SuperSeries: |
| GSE80483 |
A multi-step transcriptional and chromatin cascade underlies motor neuron programming |
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| Relations |
| BioProject |
PRJNA319165 |
| SRA |
SRP073653 |
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