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Sample GSM4227620

Query DataSets for GSM4227620

Status

Public on Dec 26, 2019

Title

Eed-ETA-rep2

Sample type

SRA

Source name

mESC

Organism

Mus musculus

Characteristics

chip antibody: none
library strategy: RNA-seq

Treatment protocol

Indicated cells were treated for 72 h with 0.5 μM 4-hydroxytamoxifen (OHT; or EtOH as vehicle) in order to delete Ring1b gene

Growth protocol

mESCs were grown on 0.1% gelatin-coated dishes in 2i/LIF-containing GMEM medium (Euroclone) supplemented with 20% fetal calf serum (Euroclone), 2 mM glutamine (Gibco), 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco), 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 50 µM ß-mercaptoethanol phosphate buffered saline (PBS; Gibco), 1000 U/ml leukemia inhibitory factor (LIF; produced in-house), and GSK3β and MEK 1/2 inhibitors (ABCR GmbH) to a final concentration of 3 mM and 1 mM, respectively

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted with the Quick-RNA™ MiniPrep extraction kit (Zymo Research) and retro-transcribed with ImProm-II™ Reverse Transcription System (Promega) according to the manufacturer’s instructions
RNA-seq was performed following SMART-seq2 protocol (Picelli et al., 2014)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Paired-end reads were aligned to the mouse reference genome mm10, or mm10 and dm6 for histone ChIP-Rx, using Bowtie v1.2.2 (Langmead et al., 2009) without allowing for multi-mapping (–m 1) and parameters -I 10 -X 1000. PCR duplicates were removed using samblaster (Faust and Hall, 2014). Ambiguous reads mapping to both mm10 and dm6 were discarded. Peaks were called using MACS2 v2.1.1 (Zhang et al., 2008) with parameters -f BAMPE --keep-dup all -m 10 30 -p 1e-10.
Reads were aligned to the mouse reference genome mm10 using STAR v2.7 without allowing multimapping reads (--outFilterMultimapNmax 1). PCR duplicates were removed using samblaster (Faust and Hall, 2014). Gene counts were calculated using featureCounts (Liao et al., 2014) with parameters -s 0 -t exon -g gene_name using Gencode M21 (GRCm38) annotation downloaded from (https://www.gencodegenes.org/mouse/). Differential expression analyses were performed using the R package DESeq2 v1.20 (Love et al., 2014) using default parameters. Log2FoldChanges and adjusted p-values were corrected using the apeglm (Zhu et al., 2019) and IHW (Ignatiadis et al., 2016) packages, respectively. Genes with an absolute log2 fold change of 1.5 and FDR < 0.05 were considered as differentially expressed
Genome_build: mm10
Supplementary_files_format_and_content: counts.tsv : raw counts, Normalized_counts.tsv : normalized counts (DESeq2), Eed_OHT-vs-Eed_ETA_diffexp_log2fc1.5_pval0.05.tsv : differential expression results

Submission date

Dec 20, 2019

Last update date

Dec 26, 2019

Contact name

Diego Pasini

E-mail(s)

lab.pasini@gmail.com

Organization name

European Institute of Oncology

Street address

Via Adamello, 16