|
| Status |
Public on Jun 05, 2014 |
| Title |
E14_EpiLC |
| Sample type |
SRA |
| |
|
| Source name |
epiblast like cells (EpiLC)
|
| Organism |
Mus musculus |
| Characteristics |
es cell line: E14 ESC
mouse strain: 129SV
growth: differentiated to EpiLCs (serum free, N2B27 based, bFGF and KOSR)
|
| Treatment protocol |
All samples were washed once with PBS and lysed directly on the plate with Trizol.
|
| Growth protocol |
All mouse ESC lines were adapted for a minimum of 5 passages to growth in serum free N2B27 based medium supplemented with MEK inhibitor PD0325901 (0.8μM) and GSK3β inhibitor CHIR99021 (3.3μM) in tissue culture dishes pretreated with 7.5μg/ml polyL-ornithine (Sigma) and 5μg/ml laminine (BD) (Hayashi et al., 2011). To induce EpiLC differentiation, cells were washed with PBS, trypsinized and strained. 200,000 to 300,000 cells per 10cm2 were plated on tissue culture dishes pretreated with 5μg/ml Fibronectin (Millipore) in N2B27 based medium supplemented with 1% KSR (Invitrogen) and 12μg/ml bFGF (Peprotech). Where indicated 20ng/ml Activin A (R&D Scientific) was added.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAs from two independent biological replicates of ESC culture and two independent biological replicates from EpiLCs were extracted with Trizol (Invitrogen), following the manufacturer's recommendations. 10 μg of total RNA were subjected to two rounds of oligo-dT purification using Dynaloligo-dT beads (Invitrogen). 100 ng of the purified RNA were fragmented with 10x fragmentation buffer (Ambion). Fragmented RNA was used for first-strand cDNA synthesis, using random hexamer primers (Invitrogen) and SuperScript II enzyme (Invitrogen). Second strand cDNA was obtained by adding RNaseH (Invitrogen) and DNA Pol I (New England Biolabs) to the first strand cDNA mix.
The resulting double-stranded cDNA was used for Illumina library preparation and sequenced with Illumina Genome Analyzer.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Mouse embryonic stem cells grown under ESC (serum free, N2B27 based 2i+LIF) or differentiated to EpiLCs (serum free, N2B27 based, bFGF and KOSR) for 48h
|
| Data processing |
RNA seq libraries were sequenced with Illumina Genome Analyzer
reads were mapped with tophat, FPKM were determined using Cufflinks
Genome_build: mm9 and refseq
Supplementary_files_format_and_content: csv files containing all FPKM
|
| |
|
| Submission date |
Mar 21, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Christa Buecker |
| E-mail(s) |
cbuecker@stanford.edu
|
| Organization name |
Stanford University
|
| Department |
Chemical and Systems Biology
|
| Lab |
Joanna Wysocka
|
| Street address |
269 Campus Drive
|
| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL11002 |
| Series (2) |
| GSE56096 |
Reorganization of enhancer patterns in transition from naïve to primed pluripotency (RNA-seq) |
| GSE56138 |
Reorganization of enhancer patterns in transition from naïve to primed pluripotency |
|
| Relations |
| BioSample |
SAMN02693804 |
| SRA |
SRX497856 |
