|Public on Dec 30, 2019
tissue: Visceral Fat
|The samples used for this study are from the red portion of the gastrocnemius muscle and visceral fat. Mice (Cre-control vs MyD88 Muscle KO) were subject to 5 weeks of wheel running followed by 8 days of restricted movement (small mouse cage; SMC).
|10-15 mg of tissue was placed in Tri reagent LS (Molecular Research Center, Cincinnati, OH, USA) and disrupted via hand-held homogenizer (Bio-Gen PRO200; Pro Scientific, Oxford, CT, USA). Chloroform was added to separate the RNA into an aqueous phase that was then precipitated using isopropanol. Extracted RNA was then washed with ethanol and suspended in nuclease-free water with EDTA. RNA was purified using TURBO DNase (AM2238) with Zymo RNA Clean and Concentrator-5 clean up. RNA integrity and quantity was determined using the EPOCH (Take3; BioTek) spectrophotometer.
Total RNA samples (100-500 ng) were hybridized with Ribo-Zero Gold to substantially deplete cytoplasmic and mitochondrial rRNA from the samples. Stranded RNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded Total RNA Library Prep Gold kit (20020598) with TruSeq RNA UD Indexes (20022371). Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (cat# 5067-5582 and 5067-5583), and average RIN was 8.3. The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (cat#KK4824). Individual libraries were normalized to 1.30 nM in preparation for Illumina sequence analysis. Sequencing libraries (1.3 nM) were chemically denatured and applied to an Illumina NovaSeq flow cell using the NovaSeq XP chemistry workflow (20021664).
|Illumina NovaSeq 6000
|To analyze RNA sequencing data, mouse GRCm38 FASTA and GTF files were downloaded from Ensembl release 96 and the reference database was created using STAR version 2.7.0f with splice junctions optimized for 50 base pair reads
Mapped reads were assigned to annotated genes in the GTF file using featureCounts v1.6.3
Differentially expressed genes were identified using a 5% false discovery rate with DESeq2 version 1.22.2
Supplementary_files_format_and_content: FeatureCount output files combined into a single matrix
|Oct 16, 2019
|Last update date
|Jul 02, 2020
|Micah J Drummond
|The University of Utah
|Physical Therapy and Athletic Training
|520 Wakara Way
|Salt Lake City
|Muscle specific MyD88-/- confers a sexual dimorphism protecting female mice from inactivity-induced fat accumulation