RNA Sequencing (RNA-Seq) and Transcriptome Analysis
Total RNA was extracted from CD11b+Ly6G+Ly6ClowPMN-MDSCs from the spleens of CIA and DBA1/J mice using Trizol Reagent (Vazyme Biotech, China). RNA sequencing was conducted using BGI Genomics (BGI, Shenzhen, China). The sequencing data were filtered with SOAPnuke (v1.5.2), and clean reads were obtained and stored in the FASTQ format. Clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was used to align the clean reads to the reference coding gene set, and the expression level of the gene was calculated using RSEM (v1.2.12). A heatmap was drawn using pheatmap (v1.0.8) according to gene expression in different samples. Differential expression analysis was performed using DESeq (v1.4.5) with a Q-value ≤ 0.05. To gain insight into the changes in phenotype, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of annotated differentially expressed genes were performed using Phyper based on the hypergeometric test. The significance levels of terms and pathways were corrected by the Q value with a rigorous threshold (Q value ≤0.05) by Bonferroni correction.