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Sample GSM1470296 Query DataSets for GSM1470296
Status Public on Jun 30, 2015
Title ESC_shHEB_rep3
Sample type SRA
Source name Embryonic stem cells
Organism Mus musculus
Characteristics strain: CGR8
passages: 18-25
activin treatment: no
Treatment protocol At day 2, human Activin A (100 ng/ml; R&D systems) was added to induce endoderm differentiation without dissociation/reaggregation.
Growth protocol Mouse embryonic stem cells (ESCs) were cultured on gelatin coated plates in the absence of feeder cells. ESC medium was prepared by supplementing knockout DMEM (Invitrogen) with 15% FBS, 1 mM glutamax, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol and 1000 units of LIF (Millipore). To inhibit TGF-beta signaling pathway, ESCs were treated with 10 µM SB431542 (Tocris Bioscience) for 24 hours. For defined embryoid body (EB) differentiation, ESCs were dissociated by TrypLE Express and cultured in serum-free defined differentiation media consisting of 75% Iscove’s modified Dulbecco’s medium and 25% Ham’s F12 media supplemented with 0.5x of both N2 and B27 (without retinoic acid), 0.05% BSA and 50 µg/ml ascorbic acid (Sigma).
Extracted molecule polyA RNA
Extraction protocol Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen), and messenger RNA (mRNA) was purified from the total RNA using Dynabeads Oligo(dT)25 (Invitrogen) according to the manufacturer's instructions.
RNA sequencing libraries were built using an approach which targets the 3′ end (3SEQ) as described previously (Beck et al., 2010). Heat sheared mRNA was reverse transcribed with the P7_oligo-dT Index primers. The P5 linker was then ligated to the free end, and the linker-ligated cDNA was size-selected for 200-350 bp fragments by 3% Nusieve agarose gels (Lonza). The selected DNA was amplified using primers P5 and P7_Index for 15 cycles, and the amplified product was second size-selected for 200-350 fragments. The purified library quality was assessed using Bioanalyzer and Qubit, and sequenced on the Illumina HiSeq 2000 with multiplexing.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Data processing Basecalls performed using CASAVA version 1.8.2
Read sequences were trimmed to remove the 3SEQ adapters and leading or trailing low-quality bases using Trimmomatic 0.32 with parameters ILLUMINACLIP 2:30:5, LEADING:3, TRAILING:3
Trimmed reads were aligned by STAR 2.3.0e to the mm9 reference genome and a reference transcriptome prepared by combining Ensembl gene annotations with ENCODE RNA-seq data from E14 mouse ESCs using Cufflinks 2.2.1; candidate splice junctions that were both non-canonical and unannotated were discarded
Reads were counted in the last 1 kb of each transcript by the featureCounts tool in Subread 1.4.5,
Transcript read counts were tested for significant differences between HEB and control shRNA by DESeq2 1.2.8, analyzing each cell/treatment group indepedently; transcripts with no reads from any library were ignored
Genome_build: mm9
Supplementary_files_format_and_content: transcripts.gtf: transcript annotations from Ensembl + ENCODE RNA-seq, used for read alignment
Supplementary_files_format_and_content: transcripts_last1000.gtf: last 1 kb of each transcript annotation, used for read counting
Supplementary_files_format_and_content: transcript_counts.tsv: read counts from each library in each transcript's last 1 kb
Supplementary_files_format_and_content: DESeq2_analysis_*.tsv: results of DESeq2 analysis, including significance test and regularized log transformation of read counts
Submission date Aug 11, 2014
Last update date May 15, 2019
Contact name Se-Jin Yoon
Phone 650-736-0278
Organization name Stanford University
Department Genetics
Street address 300 Pasteur Drive, Alway M311
City Stanford
State/province CA
ZIP/Postal code 94305
Country USA
Platform ID GPL13112
Series (2)
GSE60285 HEB associates with PRC2 and SMAD2/3 to regulate developmental fates [RNA-Seq]
GSE60286 HEB associates with PRC2 and SMAD2/3 to regulate developmental fates
BioSample SAMN02980971
SRA SRX674279

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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