Comprehensive RNA-seq Analysis of Muscle Tissue

RNA-extract and RNA-seq of muscle are conducted according to standard procedures of Majorbio with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2×150bp read length). The raw paired end reads were trimmed and quality controlled by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters. Then clean reads were separately aligned to reference genome with orientation mode using HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml) software (8 (link)). The mapped reads of each sample were assembled by StringTie (https://ccb.jhu.edu/software/stringtie/index.shtml?t=example) in a reference-based approach (9 (link)). To identify DEGs (differential expression genes) between two different samples, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. RSEM (http://deweylab.biostat.wisc.edu/rsem/) (10 (link)) was used to quantify gene abundances. Essentially, differential expression analysis was performed using the DESeq2 (11 (link))/DEGseq (12 (link))/EdgeR (13 (link)) with Q value ≤ 0.05, DEGs with |log2FC|>1 and Q value ≤ 0.05(DESeq2 or EdgeR)/Q value ≤ 0.001(DEGseq) were considered to be significantly different expressed genes. The transcriptomic sequence data have been deposited in the NCBI database (Accession No. PRJNA898816).