Bulk RNA-Seq and Bioinformatics Analysis

RNA was extracted from RASMCs of H3CIT + dsDNA stimuli group or control group by using the TRIzol^TM reagent (15596018, Invitrogen) according to the manufacturer’s instructions. The using concentration of H3CIT (32571, Cayman) and dsDNA (S1089, BD) was 0.03 μM and 300 ng/ml respectively. RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara). Then the libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co. Ltd. (Shanghai, China). FASTQ sequence data were mapped to rat reference genome NCBI Rnor6.0 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001895.5/) with TopHat v2.1.1. The reads per gene were counted by using “HTseq” (https://htseq.readthedocs.io, version 0.6.0). Sample quality metrics and raw read counts were imported into R for further processing. The DESeq2 R package⁵⁶ was used to estimate library size factors, normalize counts and perform differential expression analyses. Benjamini–Hochberg multiple testing correction was used to compute FDR, and genes were considered significantly differentially expressed at <5% FDR. Principal component analysis was performed in R using the top 1000 most variable genes, with normalized DESeq2 variance-stabilized transformation expression as input.