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Sample GSM5582353

Query DataSets for GSM5582353

Status

Public on Sep 16, 2021

Title

BglG_GFP_dox_RNAseq_rep3

Sample type

SRA

Source name

mESC_SPEN-AID_BglG-GFP

Organism

Mus musculus

Characteristics

cell line: TX1072
cell type: mESC
treatment: Dox24h
strain: BL/6J x CAST-EiJ

Treatment protocol

24 hours later, cells were exposed to doxycycline (1ug/mL). Cells were then collected 24 hours later.

Growth protocol

ES cells (TX1072) were seeded in 0.1% gelatin-coated 6-well plates (0.25*10^6 cells/well). Cells were grown in 2i + LIF serum-containing ES cell medium - DMEM (Sigma), 15% FBS (Gibco), 0.1mM β-mercaptoethanol, 1000 U/ml leukemia inhibitory factor (LIF, Chemicon), CHIR99021 (3uM), PD0325901 (1uM) in 8% CO2 37°C incubators.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted according to the manufacturer’s recommendations using RNeasy Mini Kit (Qiagen) with on-column DNAse digestion (Qiagen).
Only samples showing a RIN score above 9 were used to prepare RNAseq libraries (TruSeq). Libraries were sequenced using HiSeq2500 at PE100 settings.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

All data were mapped to the mouse genome mm10, using the BL6-EiJ / CAST SNPs from the mouse genome project (v5 SNP142)
Reads were trimmed using Trimgalore (v.0.4.4), mapped using STAR (2.5.3a, parameters:–outFilterMultimapNmax 1–outFilterMismatchNmax 999–outFilterMismatchNoverLmax 0.06–alignIntronMax 500000–alignMatesGapMax 500000–alignEndsType EndToEnd–outSAMattributes NH HI NM MD), and removed when mapping to the mitochondrial genome. Remaining reads were split by allele using SNPsplit (v.0.3.2). Allele-specific and the unassigned bam files were sorted, duplicates removed using picard (v.2.18.2, parameters: REMOVE_DUPLICATES = true ASSUME_SORTED = true) and pooled as the total reads. Quantification of expression was performed using featureCount (parameters: -p -t exon -g gene_id, -s 1 for stranded RNA-seq of in vitro cell, -s 0 for non-stranded RNA-seq of single embryo). Data were then analysed in R using DESeq2 (v.1.18.1), calculating the sizeFactor on the count of total reads and applying it to the allele-specific counts
Genome_build: mm10
Supplementary_files_format_and_content: processedData_countTable.tsv

Submission date

Sep 16, 2021

Last update date

Sep 18, 2021

Contact name

samuel collombet

E-mail(s)

[email protected]

Organization name

EMBL

Street address

Meyerholfstrasse