Mice liver tissue transcriptomics analysis
Three liver samples were procured from each of the Chow group, HFD group, and HFD+PTPS-2-2 group. Total RNA from these samples was meticulously collected, and the extracted RNA underwent assessment for purity and concentration to ensure the accuracy and reliability of the source data. Subsequent steps involved mRNA capture, fragmentation, and double-stranded cDNA synthesis. The end repair adapter, with P7 adapter (read1) sequence: AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC, and P5 adapter (read2) sequence: AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT, was employed. Following fragment sorting, PCR amplification, and stringent quality control at each experimental step, different libraries were amalgamated based on specified concentration and target off-machine data volume requirements for Illumina sequencing. The initial phase of data analysis involved evaluating the quality of sequencing data and filtering out low-quality data, contamination, and adapter sequences. The filtered sequencing Clean Data was then compared and analyzed against the reference genome. Utilizing samtools software, mpileup processing was conducted on the comparison results and the reference genome to derive potential SNV results for each sample. Subsequently, annovar software was employed for annotation. By referencing gene annotation information in the database, the occurrence position of SNV/InDel relative to the gene was determined, and the genomic distribution was statistically calculated. The direct expression level of a gene is denoted by its abundance on the gene, with higher gene abundance indicating elevated gene expression levels. Gene expression calculations utilized Htseq software, employing the FPKM (Fragments Per Kilobases per Million reads) method. Finally, a comparative analysis of gene expression levels under different experimental conditions was conducted through the distribution diagram of FPKM for all genes.