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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 25, 2024 |
| Title |
WT, N24, Rapa, rep1 |
| Sample type |
SRA |
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| Source name |
RC9
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| Organism |
Mus musculus |
| Characteristics |
cell line: RC9
cell type: embryonic stem cell
genotype: WT
treatment: Rapamycin 20 nM
timepoint: N24
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| Treatment protocol |
Rapamycin experiment: mESCs were plated on gelatin-coated plates in N2B27-2i + DMSO or N2B27-2i + 20 nM rapamycin. The following day, cells were washed with PBS and medium was exchanged to N2B27+DMSO or N2B27+ 20 nM rapamycin to induce differentiation for the indicated time.
RNAi experiment: mESCs were plated on gelatin-coated plates in N2B27-2i and transfected with FlexiTube siRNAs (Qiagen) using DharmaFECT 1 (Fisher Scientific, T-2001). The next day, cells were washed with PBS and medium was exchanged to either N2B27 without 2i to induce differentiation, or fresh N2B27-2i for the undifferentiated controls. Two siRNAs targeting FoxO1 (SI01005200, SI02694153) were used at a final concentration of 20 nM. As controls, siRNAs targeting GFP (siGFP) or scrambled siRNAs (siScr) were used.
MK-2206 experiment: mESCs were plated on gelatin-coated plates in N2B27-2i or N2B27-2i + 1 μM MK-2206. The following day, cells were washed with PBS and medium was exchanged to either N2B27 or N2B27+1 μM MK-2206 to induce differentiation for the indicated time, or to N2B27-2i or N2B27-2i+1 μM MK-2206 for the undifferentiated controls.
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| Growth protocol |
Mouse embryonic stem cells (mESCs) were cultured on gelatin-coated plates in DMEM high-glucose medium supplemented with 10% FBS, 2 mM L-Glutamine, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 10 µg/ml penicillin-streptomycin , 55 µM β-mercaptoethanol, 10 ng/ml LIF and 2i (1.5 μM PD0325901 and 3 μM CHIR99021). mESCs were cultured at 37°C and 5%CO2.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted using the ExtractMe kit (Blirt, EM15) following the manufacturer's instructions.
200-500 ng of RNA were used as starting material for library preparation with the Lexogen 3' mRNA Seq Library Prep Kit. Libraries size was measured with the Agilent High Sensitivity DNA kit, and their qPCR-based quantification was performed with the KAPA Library Quantification kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
WT_N24_Rapa_1
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| Data processing |
Fastq files were analysed with a Nextflow 23.04.1.5866 /nf-core/rnaseq v3.10.1 pipeline.
Quality control was performed using fastQC (v0.11.9), and transcripts were mapped to the mm10 assembly mouse reference genome using Salmon (v1.9.0) as a pseudoaligner and STAR (v2.7.10a) as an aligner.
DESeq2 (v1.38.3) was used to generate normalised count tables and to perform differential expression analyses (FDR-adjusted p-value ≤ 0.05; H0: log2FC = 0)
Assembly: mm10
Supplementary files format and content: comma-delimited text file include normalized counts for each Sample
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| Submission date |
Jan 17, 2024 |
| Last update date |
Jul 25, 2024 |
| Contact name |
Martin Leeb |
| E-mail(s) |
[email protected]
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| Organization name |
University of Vienna
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| Department |
Max Perutz Labs
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| Street address |
Dr.-Bohr-Gasse 9/3
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL30172 |
| Series (2) |
| GSE253473 |
FoxO transcription factors actuate the formative pluripotency specific gene expression programme [RNA-Seq] |
| GSE253480 |
FoxO transcription factors actuate the formative pluripotency specific gene expression programme |
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| Relations |
| BioSample |
SAMN39474342 |
| SRA |
SRX23264030 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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