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Sample GSM1355147 Query DataSets for GSM1355147
Status Public on Jun 05, 2014
Title Otx2-/-_tetON_Otx2_plusdox
Sample type SRA
 
Source name embryonic stem cells (ESC)
Organism Mus musculus
Characteristics es cell line: Otx2-/- ESC
mouse strain: 129SV
growth: under ESC (serum free, N2B27 based 2i+LIF) with dox treatment
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), dox treatment
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 SAMN02693803
SRA SRX497860

Supplementary file Size Download File type/resource
GSM1355147_Otx2ko_tetONOtx2_plusDox.csv.gz 403.6 Kb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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