RNA-seq Analysis of Liver and Intestine in Mice

The liver and intestine tissues (duodenum) of experimental mice were isolated and stored at -80°C until use. Then the frozen tissues (50 mg/sample) were send for RNA-extraction and RNA-seq analysis (Majorbio Bio-pharm Technology Co.,Ltd, Shanghai, China). Briefly, total RNA was extracted from different groups with three replicates. RNA-seq transcriptome library was prepared with 1 μg of total RNA using TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA). Then the paired-end RNA-seq library was sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150 bp read length). The raw paired-end reads were trimmed and quality was controlled by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle), followed by aligning to the reference genome with orientation mode using HISAT2 software (57 (link)). The mapped reads of each sample were assembled by StringTie in a reference-based approach (58 (link)). The expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. The TPM value of each DEG was shown in supplementary files. Transcript abundances were quantified using RSEM software tool (59 (link)). DEGs analysis was performed by DESeq2 with adjusted P value ≤ 0.05. KEGG pathway analysis were performed using KOBAS (http://kobas.cbi.pku.edu.cn/home.do).