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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 16, 2020 |
| Title |
Pten KO EBs-3 |
| Sample type |
SRA |
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| Source name |
embryoid bodies
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| Organism |
Mus musculus |
| Characteristics |
tissue: embryoid bodies
genotype: Pten-/-
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| Treatment protocol |
The Pten-/- ESCs and Pten-A3 mutant ESCs were generated by the CRISPR-Cas9 system. ESCs were cultured in 6-well plates and transfected with donor plasmid and constructs containing Cas9 and target sequences using lipofectamine 3000, and these were then subjected to cell sorting of GFP-positive cells after transfection for 48 h. The enriched gene-modified cell populations were cultured in 60-mm dishes for 4-5 days, and each cell clone was passaged into 24-well plates. Finally, the Pten−/− and Pten-A3 mutant cell lines were identified by sequencing, and the cells were stored in liquid nitrogen.
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| Growth protocol |
Mouse ESCs were cultured in DMEM/F12 containing GlutaMAX and sodium pyruvate (Life Technologies, 10565) with 15% fetal bovine serum (Hyclone, SH30071), nonessential amino acid solution (Life Technologies, 11140), -mercaptoethanol (Life Technologies, 31350) with 1 × 103 units/ml LIF (Millipore, ESG1106), 1 uM PD0325901 (Sigma, PZ0162), and 2.5 uM CHIR99021 (Sigma, SML1046). The mouse ESCs were grown on gelatin-coated plates and the medium was replaced every other day. The ESC cultures were passaged every 2 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
ESCs and embryoid bodies were collected, and RNA was harvested using Trizol reagent. The quality of the isolated RNA was determined with Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit). The RNA was converted into a template molecules library for sequencing on the BGISEQ-500.
Briefly, total RNA samples were ribo-depleted and then fragmented. The fragmented samples were reversed transcribed to cDNA, end-repaired and 3’ adenylated. Adaptors were ligated to the ends of these 3’ adenylated cDNA fragments. The ligation products were purified, and many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer. Denature the PCR products by heat, and the single strand DNA is cyclized by splint oligo and DNA ligase.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
BGISEQ-500 |
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| Data processing |
The raw reads were cleaned using internal software SOAPnuke (Version v1.5.2, Website: https://github.com/BGI-flexlab/SOAPnuke) by removing adapter sequences, reads in which unknown bases are more than 10%, and low quality reads.
The clean reads were stored in FASTAQ format.
We used HISAT (Version v2.0.4, Website: http://0-www-ccb-jhu-edu.brum.beds.ac.uk/software/hisat) to do the mapping step, and calculated gene expression with a software package called RSEM (Version v1.2.12, Website: http://deweylab.biostat.wisc.edu/ RSEM).
FPKM method was used in calculated expression level, and the calculated gene expression can be directly used for comparing the difference of gene expression among sample.
Genome_build: HG19
Supplementary_files_format_and_content: FPKM values for each Sample
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| Submission date |
Jul 18, 2018 |
| Last update date |
Feb 17, 2020 |
| Contact name |
Gang Lu |
| E-mail(s) |
lugang@cuhk.edu.hk
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| Organization name |
The Chinese University of Hong Kong
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| Street address |
Sha Tin
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| City |
Hong Kong |
| ZIP/Postal code |
852 |
| Country |
China |
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| Platform ID |
GPL23479 |
| Series (1) |
| GSE117280 |
RNA-seq analysis of Pten's function in regulating embryonic stem cell transition from naïve to primed pluripotent state |
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| Relations |
| BioSample |
SAMN09685448 |
| SRA |
SRX4403202 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3290398_Pten_KO_EBs-3.xlsx |
360.2 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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