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Sample GSM3399473

Query DataSets for GSM3399473

Status

Public on Sep 30, 2019

Title

RNA_RA_rep1

Sample type

SRA

Source name

Embryonic stem cells

Organism

Mus musculus

Characteristics

cell line: E14 ES cells
treatment: RA

Treatment protocol

E14 mESC cells were cultured in the presence of leukemia inhibitory factor (LIF) in order to maintain the pluripotent state of the cells. To exit from pluripotency and push the cells towards differentiation, LIF was withdrawn and retinoic acid (RA, Sigma, R2625) was added to the medium to a final concentration of 1 µM. For all experiments, 4h prior to harvest, cell culture medium was removed, cells washed twice with PBS and fresh ES medium was added containing either LIF or RA.

Growth protocol

E14 mouse embryonic stem cells (mESCs) were cultured under feeder-free conditions and routinely passaged every two days in ES medium plus LIF: Glasgow Minimum Essential Medium (Sigma-Aldrich) supplemented with 17% FBS (HycloneTM, SV30160.03, GE Healthcare), 2 mM GlutaMAXTM (Gibco), 100 U/ml Penicillin-Streptomycin (Gibco), 1x MEM Non-Essential Amino Acid Solution (Gibco), 1 mM Sodium Pyruvate (Gibco), 0.5 mM 2-Mercaptoethanol (Gibco), and recombinantly expressed LIF.

Extracted molecule

polyA RNA

Extraction protocol

2x105 low passage (< 10) E14 cells were plated per 10 cm culture dish and cultivated for 48 h in regular ES medium containing LIF. 4 h prior to harvest, medium was exchanged and cells were treated with LIF or RA as described in the treatment protocol. Cells were harvested and subjected to RNA extraction applying RNeasy Mini Kit (Qiagen) according to the manufacturers’ instructions. The experiment was performed in biological triplicates.
Sequencing libraries were generated using TruSeq® Stranded mRNA Kit (Illumina) according to manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

E14_RNA-seq_DESeq2_results.txt

Data processing

RNA-seq: Raw sequencing reads were mapped to mm10 reference genome using Burrows-Wheeler Alignment tool (BWA-MEM algorithm, PMID: 19451168), processing fastq to bam files. Mapped reads were sorted using SAMtools (PMID: 19505943) and DESeq2 (PMID: 25516281) was applied for differential gene expression analysis using ENSEMBL gene annotation (Mus_musculus.GRCm38.90).
ChIP-seq: Each ChIP library was sequenced in two separate sequencing runs. Raw sequencing reads were mapped to mm10 reference genome using STAR Alignment tool (STAR_2.5.1b_modified, PMID: 23104886), allowing a maximum intron size of 1 to adjust for non-spliced input data. Subsequently reads from the same library were merged into one file.
ATAC-seq: We processed fastq to bam files using the Burrows-Wheeler Alignment tool (BWA) (PMID: 19451168) for mapping and SAMtools (PMID: 19505943) for filtering, sorting and removing duplicates.
Genome_build: mm10
Supplementary_files_format_and_content: RNA-seq: The DESeq2 derived final data file contains all genes with an adjusted p-value below 0.1 (padj < 0.1).
Supplementary_files_format_and_content: ChIP-seq: Mapped reads were converted to bigwig coverage tracks using the bam2bigwig script (https://github.com/milospjanic/bam2bigwig).
Supplementary_files_format_and_content: ATAC-seq: We called peaks on the alignment results using MACS2 (PMID: 18798982) and converted bam files to bigwig format using the bamToBigWig tool from the bioinformatics library Gonetics (P. Benner. Gonetics. https://github.com/pbenner/gonetics).

Submission date

Sep 24, 2018

Last update date

Sep 30, 2019

Contact name

Laura V Glaser

E-mail(s)

glaser@molgen.mpg.de

Phone

+49 30 8413-1560

Organization name

Max Planck Institute for Molecular Genetics