 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 20, 2018 |
| Title |
N33_F rep1 |
| Sample type |
SRA |
| |
|
| Source name |
mouse ESCs
|
| Organism |
Mus musculus |
| Characteristics |
cell line: N33
cell type: ESC
strain: C57BL/6
culture condition: serum with feeder
|
| Treatment protocol |
ESCs were washed by PBS and trypsinized to single cells. ESCs cultured with feeders were removed off feeders twice based on their differences in the adherence to the bottom of dish.
|
| Growth protocol |
For routine feeder culture condition, ESCs were cultured under 5% CO2 at 37 °C on mitomycin C-treated MEF feeder in ES cell culture medium consisting of knockout DMEM supplemented with 20% fetal bovine serum (FBS, ES quality, Hyclone), 1,000 U/ml leukemia inhibitory factor (LIF)(ESGRO, Chemicon), 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 1 mM L-glutamine, and penicillin (100 U/ml) and streptomycin (100 µg/ml). For feeder-free culture (serum condition), plates were pre-coated with gelatin. Chemically defined serum and feeder-free culture conditions (N2B27+2i): ESCs were cultured on poly-L-ornithine and laminin-coated plates using N2B27 medium (DMEM/F12 and Neurobasal medium mixed at a ratio of 1:1, 1xN2 supplement, 1xB27 supplement, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 0.1 mM nonessential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin) supplemented with Gsk3β inhibitor (CHIR99021, 3 μM) and Mek inhibitor (PD0325901, 1 μM) and 1,000 U/ml LIF.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with RNeasy Mini Kit (Qiagen), according to manufacturer’s instructions.
The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Then the selected RNA was fragmented and copied into first strand cDNA, which was followed by second strand cDNA synthesis. These cDNA fragments then had the addition of a single 'A' base and subsequent ligation of the adapter. The products were then purified and enriched with PCR amplification. PCR yield was quantified and samples were pooled together to make a single strand DNA circle (ssDNA circle), which gave the final library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
BGISEQ-500 |
| |
|
| Data processing |
Basecalls performed using the software Zebra call
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using Bowtie2.
Fragments Per Kilobase per Million is calculated to represent the gene expression level. FPKM = numFragments / ( geneLength/1000 * totalNumFragments /1,000,000). numFragments - number of fragments mapped to a gene sequence. geneLength - length of the gene sequence. totalNumFragments - total number of mapped fragments of a sample.
Functional enrichment (GO annotation, KEGG) of gene sets with different expression patterns was performed using clusterProfiler. The heat maps were drawn by the function ‘pheatmap’ of R packages ‘pheatmap’ and correlation coefficients were calculation by the function ‘cor’ in R. Scatter plots were generated using the ‘ggplot2’ package to graphically reveal genes that differ significantly between two samples.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited FPKM files include FPKM values for each Sample.
|
| |
|
| Submission date |
Jan 19, 2018 |
| Last update date |
Jan 24, 2018 |
| Contact name |
Renpeng Guo |
| Organization name |
Nankai University
|
| Street address |
Weijin Road 94th, Nankai District, Tianjin, China
|
| City |
Tianjin |
| ZIP/Postal code |
300071 |
| Country |
China |
| |
|
| Platform ID |
GPL23479 |
| Series (1) |
| GSE109418 |
Transcriptome profiles of mouse embryonic stem cells cultured in different conditions by RNA-seq analysis |
|
| Relations |
| BioSample |
SAMN08379126 |
| SRA |
SRX3591583 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2942635_N33_F_rep1.gene.fpkm.txt.gz |
1.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
