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Sample GSM4697828

Query DataSets for GSM4697828

Status

Public on Jun 23, 2021

Title

ActD6

Sample type

SRA

Source name

RAW264.7

Organism

Mus musculus

Characteristics

cell line: RAW264.7
treatment: ActD, 6h

Treatment protocol

Transcription was inhibited by adding 10 µg/ml Actinomycin D (ApplicChem, A1489,0005) for 0, 2, 4 and 6 h.

Growth protocol

RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Biochrome), 2 mM L-Glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all PAN Biotech) at 37°C in a humidified atmosphere with 5% CO2. Every two to three days, cells were carefully detached by scraping in warm 1x PBS and diluted 1:10. On the day before experiments, cells were seeded at a density of 2.18 x 10^5 cells/cm2.

Extracted molecule

cytoplasmic RNA

Extraction protocol

RNA was extracted with the GeneMatrix universal RNA purification kit (Roboklon).
After depletion of rRNA, sequencing libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit by the Cell Networks Deep Sequencing Core Facility of Heidelberg University.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Reads were mapped with STAR v2.5.3a (Dobin et al., Bioinformatics 2012) to a reference of the mouse genome (mm10), providing the basic set of Gencode VM18 as downloaded from the UCSC Genome Browser wgEncodeGencodeBasicVM18 table as transcript annotations, allowing up to 2 mismatches and chimeric read detection with a minimum of 10 nt per segment (--chimSegmentMin 10).
Read counts were summarized at the gene level with the featureCounts function of the subread package v1.6.3 (Liao et al., Bioinformatics 2014), requiring that a read is completely contained within an exon to be counted.
Circular RNAs were detected with the CIRCexplorer2 v2.3.5 functions parse and annotate (Zhang et al., Genome Research 2016).
For normalization, read counts were subsequently divided by the sum of all reads spanning circRNA junctions in the corresponding sample.
Linear regression was performed on ln-transformed read counts. Half-lives were calculated from the slope of the regression line with the equation: ln(2) / -slope.
Genome_build: mm10
Supplementary_files_format_and_content: csv file containing raw read counts per gene, expression values after normalization to circRNA junction reads, half-lives in hours and the coefficient of determination (R2) of the linear regression used for determination of half-lives. The 6 h time-point was omitted for the calculation of half-lives, because prolonged exposure to ActD apparently inhibited decay.

Submission date

Jul 27, 2020

Last update date

Jun 23, 2021

Contact name

Johanna Daniela Schott

E-mail(s)

johanna.schott@medma.uni-heidelberg.de

Organization name

Heidelberg University

Department

Mannheim Institute for Innate Immunoscience