Transcriptome analysis using microarrays

RNA was isolated from sorted or cultured ILC2s as previously described (79). Briefly, total RNA was isolated from sorted cells flash-frozen in PBS and stored at −80°C before RNA extraction. QIAzol Lysis Reagent (Qiagen) was added to the cells, and RNA was isolated and purified using the RNeasy Micro Kit (Qiagen). Total RNA was amplified using the GeneChip WT Pico Kit (Thermo Fisher Scientific), generating biotinylated sense-strand DNA targets. Labeled samples were hybridized to human Clariom D Pico arrays (Thermo Fisher Scientific). Washing and staining were performed with GeneChip Fluidics Station 450, and scanning was conducted using GeneChip Scanner 3000 (both Thermo Fisher Scientific). All cell populations were analyzed in triplicate; data analysis was performed in RStudio (v1.1.383). Raw data were normalized using the RMA algorithm implemented in the limma Bioconductor R package (80). Adjusted P values were calculated with a moderated t test corrected for multiple testing using the Benjamini-Hochberg method. Data were visualized using glimma and pheatmap R packages (81).