|
| Status |
Public on Jun 30, 2023 |
| Title |
TRIM28 mKO Sham1 |
| Sample type |
SRA |
| |
|
| Source name |
plantaris muscle
|
| Organism |
Mus musculus |
| Characteristics |
tissue: plantaris muscle
genotype: Trim28 Knockout
treatment: Sham
|
| 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
|
| City |
Madison |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE236262 |
A Novel Method for Visualizing In-Vivo Rates of Protein Degradation Provides Insight into how TRIM28 Regulates Muscle Size |
|
| Relations |
| BioSample |
SAMN36176564 |
| SRA |
SRX20852279 |
