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Sample GSM4548774

Query DataSets for GSM4548774

Status

Public on Jul 06, 2020

Title

treatment_mrt_stimulation_il17_rep1

Sample type

SRA

Source name

ST2 treatment_mrt_stimulation_il17

Organism

Mus musculus

Characteristics

cell line: ST2

Treatment protocol

For the RNA sequencing experiment, ST2 cells were grown in 6 well plates, washed and stimulated with IL-17 (500 ng/ml) in the presence or absence of MRT67307 (2 μM) in DMEM supplemented with 0.5% FCS.

Growth protocol

Cells were cultivated in DMEM supplemented with 10% fetal calf serum (FCS) (Gibco), 100 U/ml penicillin (BB Pharma), 100 μg/ml streptomycin (Sigma Aldrich), 40 μg/ml gentamicin (Sandoz).

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from the cells using RNeasy Mini Kit (Qiagen) with on column DNAse treatment exactly according the manufacturers protocol. The quality and quantity of isolated RNA was evaluated using Nanodrop and TapeStation 2200 (Agilent Technologies). The ensuing RNA sequencing analysis was based on three independent experiments.
For each sample, 1 ug of total RNA (RIN >7.0, rRNA ratio (28S:18S rRNA) >1.0) was sent to Macrogen Inc. for library preparation and sequencing. Briefly, first TruSeq RNA stranded library was generated and then sequencing was performed on the Illumina NovaSeq6000 with 151bp paired-ends configuration, with about 30M reads per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

RNA was quantified at the transcript-level using Salmon (0.13.1) (Patro et al, 2017). First, a reference mouse transcriptome from Gencode M21(GRCm38.p6) was created by combining the sequence of protein-coding genes and long non-coding RNAs. Then, the transcriptome was indexed using Salmon index with parameter `–gencode`. Salmon quant was then run for each sample using parameters `–gcBias`, `–validateMappings`, and `–allowDovetail`.
Transcript-level estimates from Salmon were summarized into gene level estimates using tximport (Soneson et al, 2015); all genes with at least 5 reads in at least 1 sample were retained for downstream analysis; with these parameters 15,935 genes were obtained. Differential gene expression analysis was performed using DESeq2 (Love et al, 2014), using independentFiltering and using the Benjamini-Hochberg procedure to adjust p-values. Genes with adjusted p-value less than 0.01 are reported as statistically significant.

Submission date

May 12, 2020

Last update date

Jul 06, 2020

Contact name

Peter Draber

E-mail(s)

peter.draber@lf1.cuni.cz

Organization name

First Faculty of Medicine, Charles University

Department

BIOCEV