Nanodrop nd 2000 system

We selected three groups of HaCaT cells for RNA sequencing: untreated cells, 600 μM H₂O₂-induced cells, and H₂O₂-induced cells pre-treated with 40 μM ISO (pre-treatment with ISO markedly increased cell viability in a dose-dependent manner versus that observed for cells treated with H₂O₂ alone. 40 μM ISO was significantly different to 20 μM but there was no difference with 60 μM. The cells pre-treated with 40 μM ISO were used as the protection group). Total RNA was extracted from the cells using TRIzol reagent according to the manufacturer’s instructions. The absorbance ratio at 260–280 nm of the RNA samples was measured with a Nanodrop ND-2000 system (Thermo Fisher Scientific) and the RNA integrity number was determined using an Agilent Bioanalyzer 4150 system (Agilent Technologies). Only high-quality samples according to assessments of RNA purity, concentration, and integrity were used for library construction.

Total RNA was extracted from the lung using TRIzol reagent according to the manufacturer’s instructions (Magen, China). The quantity and quality of RNA samples were examined using a Nanodrop ND-2000 system (Thermo Scientific, USA) and an Agilent Bioanalyzer 4150 system (Agilent Technologies, CA, USA). Paired-end libraries were prepared using an ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) following the manufacturer’s instructions. The mRNA generated from 1 μg total RNA was purified using oligo magnetic beads, followed by fragmentation using divalent cations at elevated temperatures in ABclonal first strand synthesis reaction buffer. Then, the first-strand cDNAs were synthesized using random hexamer primers and reverse transcriptase, followed by second-strand cDNA synthesis. The double-stranded cDNAs were adapter-ligated for PCR amplification, followed by purification using an AMPure XP system. After quality control on an Agilent Bioanalyzer 4150 system, the library was sequenced on an Illumina Novaseq 6000 or MGISEQ-T7 instrument. The 150 bp paired-end reads were generated. The raw data were analyzed using an in-house pipeline from Shanghai Applied Protein Technology (China). The genes with adjusted P value < 0.05 and |log2 fold change|> 1 were identified as DEGs. Heatmap was generated using Z-scores.

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 B05.10 strain was cultured in YEPD medium at 25°C for 24 h, then half was treated with PA at a final concentration of 0.2 μL/mL and the other half was treated with H₂O at 25°C for 4 h. Total RNA was extracted from the treated mycelia using the TRIzol reagent (Magen, Guangzhou, China) by following the manufacturer’s protocol. The quality and concentration of the RNA samples were measured with the NanoDrop ND-2000 system (Thermo Scientific, USA). High-throughput transcriptome sequencing (RNA-seq) was performed with Illumina NovaSeq 6000 platform, from which 200-bp paired-end reads were obtained.
Differentially expressed genes (DEGs) between the PA-treated and H₂O-treated B05.10 were identified using DESeq2. A log₂ fold change of greater than 2 or less than −2 with a P value of <0.05 was considered differential expression. To explore the functions of the DEGs, gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using clusterProfiler R. The sequencing and sequence analysis were conducted by APTBIO Co., Ltd. (Shanghai, China).