RNA sequencing of TA skeletal muscle of mice with conditional ablation of dystrophin within the myofiber

We generated and analyzed a conditional Dystrophin flox52/Y: human alpha-skeletal actin muscle knockout (Dmd flox52/Y: HSA mKO) model by targeting exon 52 deletion in the Dystrophin gene to study the consequences of dystrophin loss in skeletal muscle lineages. The generated DMD mouse model ablates dystrophin protein expression after mating with a Cre recombinase transgenic mouse under the control of HSA promoter element that is restricted to the skeletal muscle. The resulting Dmd mKO mice have been assessed using histopathological, phenotypical, functional and biochemical assays based on TRET-NMD standard operating protocols (SOPs) for mdx mouse models. Phenotypic analysis of these conditional Dmd mKO mice revealed a significant decline in locomotor activity and reduced muscle force, motor and muscular function. The histochemical analysis revealed an increase in centralized myonuclei and fibrotic area similar to mdx mice. Immunoassays including western blot and immunohistochemistry confirmed low expression levels of dystrophin in skeletal muscles of Dmd mKO mice. Bulk RNA sequencing analysis revealed that dystrophin loss in myofiber significantly disrupted the expression of cytokines and extracellular matrix genes. To investigate the molecular changes of myofiber dystophin loss in Dmd flox52/Y: HSA mKO mice Five mice per group were used for RNA-sequencing and gene expression analysis. Total RNA was extracted from tibialis anterior (TA) muscle using Trizol reagent. Quality of RNA was assessed by Nanodrop and Bioanalyzer. Purified RNA was used to generate cDNA. Duplicates of cDNA samples were amplified on the QuantStudio 3 Real-Time PCR system using PowerUp SYBR Green Master Mix. RNA sequencing library construction and next-generation sequencing were performed by Novogene. Purified RNA from TA muscles of Dmd flox52/Y: HSA mKO and Dmd flox52/Y mice was measured by Agilent Bioanalyzer. High quality total RNA with RNA integrity value higher than 7 was used for library construction. After mRNA isolation, RNA was fragmented in First Strand synthesis buffer. Then, first-strand CDNA synthesis using reverse transcriptase and random hexamer primer was performed. Synthesis of cDNA using second-strand master mix, end-repair and dA-tailing were performed. The library was amplified and purified using AMPure beads. The library size and mass were evaluated and quantititative PCR was performed to validate the libraries. Barcoded samples were pooled equimolarly for simultanoeus sequencing. The quality of RNA-sequencing data was assessed for downstream analysis. Low-quality sequences and reads were removed. The expected read counts and FPKM were used for further analysis.