Effects of Pten knockout in dystrophic skeletal muscles of mdx mouse

Analysis of skeletal muscles with specific knockout (KO) of Phosphatase and tensin homolog (Pten) gene in an animal model of DMD (mdx mice). Pten knockout alleviates myofiber degeneration and restores muscle function in mdx mice. Total RNA was isolated from ~30 mg diaphragm muscles from experimental Pten knockout and control D2.B10-mdx mice (10-week old) using TRIzol reagent according to the manufacturer’s instructions. RNA was quantitated at the Qubit fluorometer and quality-controlled at an Agilent 2100 Bioanalyzer. A complementary DNA library was constructed using polyA selected RNA, and sequencing was performed according to the Illumina HiSeq standard protocol (high-throughput, paired-end 150bp fragment sequencing). Raw reads from RNA-seq libraries are filtered to remove reads containing adapters or reads of low quality. After filtering, statistics analysis of data production and quality was performed to confirm the sequencing quality. Reference genome and gene annotation files were downloaded from a genome website browser (NCBI/UCSC/Ensembl). TopHat2 was used for mapping the filtered reads to the reference (Mus Musculus, GRCm38/mm10) genome. For the quantification of gene expression level, HTSeq V0.6.1 was used to analyze the read numbers mapped for each gene. The Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of each gene was calculated based on the gene read counts mapped to genes or exons. A differential expression analysis was performed using the DESeq2 R/EdgeR R package with the threshold of significance set as adjusted p < 0.05.