 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 25, 2024 |
| Title |
FoxO3, Pten KO N24, rep2,ChIP |
| Sample type |
SRA |
| |
|
| Source name |
RC9
|
| Organism |
Mus musculus |
| Characteristics |
cell line: RC9
cell type: embryonic stem cell
genotype: Pten KO
timepoint: N24
chip antibody: FoxO3
|
| Treatment protocol |
mESCs were plated on gelatin-coated plates at a final density of 1 × 104 cells/cm2 in N2B27 medium (1:1 mix of DMEM/F12 ) and Neurobasal medium supplemented with N2, B27 Serum-Free Supplement, 2 mM L-Glutamine, 0.1 mM NEAA, 10 µg/ml penicillin-streptomycin, 55 µM β-mercaptoethanol) and 2i (1 μM PD0325901 and 3 μM CHIR99021). The following day, cells were washed with PBS and medium was exchanged to either N2B27 without 2i to induce differentiation for the indicated time, or to fresh N2B27-2i for the undifferentiated controls.
|
| 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.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
mESCs were fixed with 1% formaldehyde for 10 minutes, and cross-linking was then quenched with glycine. After cell lysis, chromatin was sonicated in Bioruptor® Pico Tubes (diagenode).
ChIP-seq libraries were prepared using the Ligation Sequencing kit and the Unique NEB adaptors from 1-50 ng of DNA as starting material. Libraries size was measured with the Agilent High Sensitivity DNA kit (median of 350-450 bp), and their qPCR-based quantification was performed with the KAPA Library Quantification kit.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Quality control of fastq files was performed using fastQC (v0.11.9) before and after trimming the sequencing adapter fragments with trim-galore (v0.6.7) and cutadapt (v3.5). Additional 2 bp at the 3’ end were removed with the parameters --three_prime_clip_R1 2 (--three_prime_clip_R2 2 in case of paired end reads).
The trimmed reads were aligned to the mm10 assembly mouse reference genome with bowtie2 (v2.4.4).
The obtained sam files were converted to bam files with samtools (v1.13), and uniquely mapping reads were extracted by removing duplicate reads (as potential PCR artefacts) with samtools markdup.
Peak calling was performed on bam files with macs2 (v2.2.7.1) on combined ChIP duplicates, using all Input samples as control files.
Potential artifactual regions listed in the mm10 blacklist were removed from the obtained bed files using bedtools (v2.31.0).
Assembly: mm10
Supplementary files format and content: bed
Supplementary files format and content: peaks
|
| |
|
| Submission date |
Jan 17, 2024 |
| Last update date |
Jul 25, 2024 |
| Contact name |
Martin Leeb |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Vienna
|
| Department |
Max Perutz Labs
|
| Street address |
Dr.-Bohr-Gasse 9/3
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE253479 |
FoxO transcription factors actuate the formative pluripotency specific gene expression programme [ChIP-Seq] |
| GSE253480 |
FoxO transcription factors actuate the formative pluripotency specific gene expression programme |
|
| Relations |
| BioSample |
SAMN39474799 |
| SRA |
SRX23264983 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
