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| Status |
Public on Jul 23, 2024 |
| Title |
Transcriptomic and epigenomic profiling of Tet TKO neural stem cells to study NSC specification and multipotency |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
Genome binding/occupancy profiling by high throughput sequencing
Methylation profiling by high throughput sequencing
Other
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| Summary |
In this study we: (1) identify genes deregulated in wildtype (WT) and Tet triple knockout (TKO) mouse embryonic stem cells (ESC) and neural stem cells (NSC). (2) Profile genome-wide changes in DNA methylation in WT and TKO ESCs and NSCs. (3) Map histone marks H3K4me1 and H3K27ac in WT and TKO ESCs and NSCs. (4) Identify promoter-interacting chromatin loops in WT and TKO NSCs.
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| Overall design |
We investigated the role of TET DNA dioxygenases in regulation of neural stem cell (NSC) specification from mouse embryonic stem cells (ESC) using Tet wildtype (WT) and triple knockout (TKO) ESCs in a NSC differentiation model. Analysis and integration of RNA-seq, WGBS, and CUT&Tag revealed critical roles for TET enzymes in establishment of NSC gene expression, gliogenic competence, and enhancer activation. For all omics experiments, Tet-WT and Tet-TKO ESCs were harvested after pre-plating for 45 minutes to remove feeder cells. Tet-WT and Tet-TKO NSCs were harvested on day 6 (passage 1) of NSC culture. RNA-seq: RNA was extracted from three independent Tet-WT and two independent Tet-TKO ESC and NSC lines (Qiagen RNeasy mini kit). Library preparation and RNA-seq were performed by Novogene (https://en.novogene.com/) on an Illumina Novoseq 6000 platform and 20-30 million reads were generated per sample. WGBS: DNA was extracted from one clone of Tet-WT and Tet-TKO ESC and one clone of Tet-WT and Tet-TKO NSC (Quick-DNA miniprep kit - Zymo, D3024) and subjected to WGBS analysis at BGI genomics company (https://en.genomics.cn/). Lamda DNA spike-in confirmed the efficiency of bisulfite conversion (>99.0%). CUT&Tag: Two clones each of Tet-WT and Tet-TKO ESCs and NSCs were used to profile H3K4me1 and H3K27ac. Cells were crosslinked, bound to Con-A beads, incubated with primary antibody and treated with pre-loaded pA-Tn5 adapter complex. Libraries were prepared with NEBNext HiFi 2x PCR Master mix and sent for 75-bp paired-end sequencing on an Illumina NextSeq 500 platform at the Einstein Epigenomics core following their protocols. Promoter-capture HiC: Cells were crosslinked with 4% PFA and then library preparation, promoter-capture, and sequencing were carried out by Arima Genomics. Libraries were sequenced to a depth of 170-190 million raw read-pairs. Details of RNA-seq, WGBS, CUT&Tag, and promoter-capture HiC procedure and data analyses are described in detail in the methods section of the manuscript.
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| Contributor(s) |
Dawlaty MM, MacArthur IC |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Nov 29, 2023 |
| Last update date |
Jul 26, 2024 |
| Contact name |
Meelad M Dawlaty |
| E-mail(s) |
meelad.dawlaty@einsteinmed.org
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| Phone |
718-678-1224
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| Organization name |
Albert Einstein College of Medicine
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| Department |
Genetics
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| Lab |
Dawlaty Lab
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| Street address |
1301 Morris Park Ave, Price 419, Bronx
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platforms (3) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
| GPL28457 |
DNBSEQ-G400 (Mus musculus) |
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| Samples (36)
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| Relations |
| BioProject |
PRJNA1046471 |
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