Pnpla2 knockout in muscle stem cells

Analysis of skeletal muscle satellite cells with specific knockout (KO) of Patatin-like phospholipase domain containing protein 2 (Pnpla2) gene in mouse (named Pnpla2PKO). Pnpla2 knockout disrupts lipid droplet catabolism in satellite cells and causes energy insufficiency and oxidative stress, accompanied by global lipid remodeling, which eventually impedes their expansion and fusion, leading to impairments in muscle regeneration. Overall design: FACS (fluorescence activated cell sorting)-isolated satellite cells from the experimental Pnpla2PKO and wild type control hind limb muscles were cultured for 7 days in growth medium and switched to DMEM with 2% horse serum for 16 h. Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. RNA quality analysis was performed by Agarose Gel Electrophoresis and Agilent 2100 Bioanalyzer. A complementary DNA library was then constructed using poly(A) 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 with low quality. 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 genome (Mus Musculus, GRCm38/mm10). 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.