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Sample GSM7523802

Query DataSets for GSM7523802

Status

Public on Jun 30, 2023

Title

TRIM28 mKO MTE3

Sample type

SRA

Source name

plantaris muscle

Organism

Mus musculus

Characteristics

tissue: plantaris muscle
genotype: Trim28 Knockout
treatment: MTE

Treatment protocol

Mechanical overload via myotenectomy (MTE) or a sham surgery as the control condition

Growth protocol

Mice of 9-12 weeks of age

Extracted molecule

total RNA

Extraction protocol

Frozen PLT muscles were pulverized into a fine powder using an RNase-treated and liquid nitrogen-chilled mortar and pestle. The resulting powder for each sample was collected, weighed, and placed in 0.5 ml of ice-cold TRIzol. Samples were then homogenized with a Polytron (PT 1200 E) for 20 s and then centrifuged at 6,000 x G for 1 min to remove bubbles and confirm complete homogenization. Total RNA was isolated with a PureLink RNA Mini Kit according to the manufacturer’s instructions. The purity and integrity of RNA samples were checked by the ratio of A260/A280 and of 28S/18S rRNA, respectively, and then confirmed by the University of Wisconsin-Madison Biotechnology Center using the NanoDrop One Spectrophotometer and Agilent 2100 Bioanalyzer.
Total RNA libraries were prepared from samples that met the TruSeq® Stranded Total RNA Sample Preparation Guide (15031048 E) input guidelines using the Illumina® TruSeq® Stranded Total (Gold) RNA Sample Preparation kit. For each library preparation, cytoplasmic & mitochondrial ribosomal RNA was removed using biotinylated target-specific oligos combined with paramagnetic beads tagged with streptavidin. Following purification, the reduced RNA was fragmented using divalent cations under elevated temperature. Fragmented RNA was copied into first-stranded cDNA using SuperScript II Reverse Transcriptase and random primers. Second-strand cDNA was synthesized using a modified dNTP mix (dTTP replaced with dUTP), DNA Polymerase I, and RNase H. Double-stranded cDNA was cleaned up with AMPure XP Beads (1.8X). The cDNA products were incubated with Klenow DNA Polymerase to add a single ‘A’ nucleotide to the 3’ end of the blunt DNA fragments. Unique dual indexes (UDI) were ligated to the DNA fragments and cleaned up with two rounds of AMPure XP beads (0.8X). Adapter ligated DNA was amplified by PCR and cleaned up with AMPure XP beads (0.8X). Final libraries were assessed for size and quantity using an Agilent DNA1000 chip and Qubit® dsDNA HS Assay Kit, respectively. Libraries were standardized to 2 nM. Paired-end 150 bp sequencing was performed, using standard SBS chemistry on an Illumina NovaSeq6000 sequencer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Raw reads contained in FASTQ files from our sequencing experiment were trimmed of adapters and poorly called bases using a Seqtk-trimfq algorithm, and were then mapped to the mouse genome (Ensemble GRCm.38) using STAR-RSEM pipeline executed in MobaXterm.
Between 137 and 444 million reads were quantified per sample. Differential gene expression analysis was performed using the EdgeR pipeline [80] implemented in R-studio using the raw reads normalized by the TMM method as the input values for each gene [81].
The differential gene expression outputs for a given comparison was FDR-corrected by EdgeR using Benjamini-Hochberg procedure.
Two-way ANOVA analysis of the data was performed in Perseus [82]
Assembly: GRCm38

Submission date

Jun 30, 2023

Last update date

Jun 30, 2023

Contact name

Troy A Hornberger

E-mail(s)

thornb1@svm.vetmed.wisc.edu

Organization name

University of Wisconsin - Madison

Street address

2015 Linden Drive