RNA sequencing of HTR-8/SVneo cells

HTR-8/SVneo cells, subjected to high glucose conditions and treated with the PLD inhibitor VU0359595, were harvested for RNA sequencing. Under these high glucose conditions, with a glucose concentration of 25 mM being maintained, VU0359595 was introduced to achieve a final concentration of 10 μM. After drug treatment, cell samples were collected and total RNA was extracted from the cells using Trizol reagent according to the manufacturer's instructions. The extracted RNA was evaluated for quality using a nanodrop spectrophotometer and agarose gel electrophoresis. The RNA integrity (RIN value) was assessed using an Agilent 2100 bioanalyzer to ensure that the RNA was suitable for subsequent sequencing experiments. Subsequently, the Illumina TruSeq RNA Sample Preparation Kit was used to construct an RNA-Seq library according to the manufacturer's instructions, and next-generation high-throughput sequencing was performed using the Illumina sequencing platform. After obtaining the raw sequencing data, the quality of the raw sequencing data was controlled using FastQC software, and low-quality sequences were removed. Then, the clean RNA seq data was compared with the human reference genome (GRCh38) using the HISAT2 software. After the comparison was completed, the reads of each RNA were calculated using the featureCounts or HTSeq-count tools.