Agilent bioanalyzer 4150 system

According to the manufacturer’s instructions, total RNA was extracted from ovine LTL using TRIzol^® reagent (Merck KGaA, Darmstadt, Germany). RNA samples were quantified on the basis of the A260/A280 absorbance ratio using a Nanodrop ND-2000 system (Thermo Scientific, Waltham, MA, USA), and the RIN of the RNA was calculated using an Agilent Bioanalyzer 4150 system (Agilent Technologies, Santa Clara, CA, USA). The PCR products were purified (AMPure XP system), and the quality of the libraries was evaluated via an Agilent Bioanalyzer 4150 system. Finally, paired-end reads of 150 bp were generated by sequencing the library preparations using an Illumina Novaseq 6000. Bioinformatic analysis was performed using the data generated via the Illumina (or BGI) platform.

Total RNA was extracted from the liver tissues of mice in WT, db/db, and db/db + ICS II (40 mg·kg⁻¹) groups using TRIzol buffer on the basis of experimental protocol. RNA samples were detected based on the A260/A280 absorbance ratio with a Nanodrop ND-2000 system (Thermo Scientific, Waltham, MA, USA), and the RIN of RNA was determined by an Agilent Bioanalyzer 4150 system (Agilent Technologies, Santa Clara, CA, USA). Only qualified samples will be used for library construction. Sequencing was performed with an Illumina Novaseq 6000 /MGISEQ-T7 instrument. The data generated from Illumina/BGI platform were used for bioinformatics analysis. FeatureCounts (http://subread.sourceforge.net/, accessed on 7 May 2021) was used to count the reads numbers mapped to each gene. Then, the FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Gene expression with a fold change (FC) greater than 1.5 and P-value less than 0.05 were identified as differentially expressed genes (DEGs) in this study. The enrichment of pathways and functional processing were formed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and annotations of Gene Ontology (GO) (http://www.genome.jp/kegg/pathway.html, accessed on 7 May 2021).

The total RNA is isolated in BMDMs of Sirt3^+/+ and Sirt3^−/− treated with C16:0 + MSU in triplicate. Transcriptome sequencing and subsequent bio-informatics analysis were carried out by the Applied Protein Technology Co., Ltd. (Shanghai, China). The mRNA was purified from 1 μg total RNA using oligo (dT) magnetic beads followed by fragmentation in the ABclonal First Strand Synthesis Reaction Buffer. Subsequently, using mRNA fragments as templates, the first strand of cDNA is synthesized using random primers and Reverse Transcriptase (RNase H), followed by the second strand of cDNA synthesis using DNA polymerase I, RNAseH, buffer, and dNTPs. The synthesized double-stranded cDNA fragments are ligated to the linker sequence for PCR amplification. The PCR product was purified and library quality was evaluated using Agilent Bioanalyzer 4150 system. Finally, the library preparations were sequenced on an Illumina Novaseq 6000 (or MGISEQ-T7), and 150 bp paired-end reads were generated. The data generated from Illumina (or BGI) platform were used for bioinformatics analysis. Differential expression genes (DEGs) analysis was performed using the DESeq2, DEGs with | log2FC |> 1 represent upregulation, DEGs with | log2FC |< − 1 mean downregulation, and Padj < 0.05 were considered to be significantly differentially expressed genes.