GSE158912
Cpt2 knockout in muscle stem cells
Analysis of skeletal muscle satellite cells with specific knockout (KO) of Carnitine Palmitoyltransferase 2 (Cpt2) gene in mouse (named Cpt2PKO). Cpt2 knockout disrupts fatty acid oxidation in satellite cells and causes energy insufficiency and alteration of protein acetylation, which eventually impedes their expansion and differentiation, leading to impairments in muscle regeneration. Overall design: FACS (fluorescence activated cell sorting)-isolated satellite cells from the experimental Cpt2PKO and wild type control hind limb muscles were cultured for 7 days in growth medium. 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.