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Sample GSM5173458

Query DataSets for GSM5173458

Status

Public on May 12, 2022

Title

Muscle_Sirt6-mKO_1

Sample type

SRA

Source name

Muscle tissue

Organism

Mus musculus

Characteristics

strain: C57BL/6
genotype: mdx, Sirt6-mKO

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from muscle tissue using the miRNeasy micro Kit (Qiagen) combined with on-column DNase digestion (DNase-Free DNase Set, Qiagen)
300 ng of total RNA was used as input for VAHTS Stranded mRNA-seq Library preparation following manufacture’s protocol (Vazyme)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Raw reads were assessed for quality, adapter content and duplication rates with FastQC 0.11.8 (Andrews S. 2010, FastQC: a quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc).
Trimmomatic version 0.38 was employed to trim reads after a quality drop below a mean of Q15 in a window of 5 nucleotides (Bolger et al., Trimmomatic: a flexible trimmer for Illumina sequence data). Only reads longer than 15 nucleotides were cleared for further analyses.
Trimmed and filtered reads were aligned versus the Ensembl mouse genome version mm10 (GRCm38.p5) using STAR 2.6.1d with parameters “--outFilterMismatchNoverLmax 0.1 --outFilterScoreMinOverLread 0.9 --outFilterMatchNminOverLread 0.9 --outFilterMatchNmin 20 --alignMatesGapMax 2000 --alignIntronMax 200000” (Dobin et al., STAR: ultrafast universal RNA-seq aligner).
The number of reads aligning to genes was counted with featureCounts 1.6.3 from the Subread package (Liao et al., featureCounts: an efficient general purpose program for assigning sequence reads to genomic features). Only reads mapping at least partially inside exons were admitted and aggregated per gene. Reads overlapping multiple genes or aligning to multiple regions were excluded.
Differentially expressed genes were identified using DESeq2 version 1.18.1 (Love et al., Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2).
The Ensemble annotation was enriched with UniProt data based on Ensembl gene identifiers (Activities at the Universal Protein Resource (UniProt)).
Genome_build: mm10 / GRCm38
Supplementary_files_format_and_content: Tab-delimited matrix with DESeq normalized gene counts.

Submission date

Mar 16, 2021

Last update date

May 12, 2022

Contact name

Carsten Kuenne

E-mail(s)

[email protected]

Organization name

Max Planck Institute for Heart and Lung Research

Department

Bioinformatics