RNA sequencing

CD11c-Cre and CD11c-Cre IL-33^fl/fl mice were injected (intraperitoneally) with 2 × 10⁶ Flt3L-producing EL4 cells, and MLNs were harvested 7 days later to prepare single-cell suspensions and electronically sorted out CD45⁺B220⁻CD3⁻NK1.1⁻CD64⁻CD11c⁺MHCII⁺CD103⁺ cells. RNA was isolated from sorted cells using the Qiagen RNAeasy Plus kit (Qiagen, Hilden, Germany), and library was prepared with 10 ng of RNA using Clontech SMART-seq ultralow input complementary DNA (cDNA) synthesis (Takara, Mountain View, CA) and Nextera XT library prep (Illumina, San Diego, CA) kits. The samples were then loaded onto platform for a single read of 75 cycles at the depth of 95 million reads. Sequencing reads were trimmed for quality (Phred score < 33) and to remove adapter sequences using the Trimmomatic software (30). High-quality reads were aligned to the GRCm38.p6/mm10 reference genome using the STAR aligner (31) and quantified to genes with HTSeq-count. Differentially expressed genes were obtained using the DESeq2 tool in the R statistical software, and GSEA was assessed using the GSEA R tool.