RNA sequencing and analysis

RNA was extracted from microglia purified by magnetic bead cell sorting. Briefly, retina and RPE-choroid-sclera complex single-cell suspensions were prepared as described above, and CD11b enrichment was performed by magnetic bead cell sorting according to the manufacturer’s instructions (Miltenyi Biotech). Total RNA from sorted cells was extracted using TRIzol reagent following the manufacturer’s instructions. RNA samples were sent to the Beijing Genomics Institute (Shenzhen, China) for quantification, cDNA library preparation, RNA sequencing, and data analysis. Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, US). After the library was qualified, it was sequenced by an Illumina NovaSeq 6000. FeatureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene. Then, the FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene, which normalizes gene expression by considering the effect of sequencing depth and gene transcript length at the same time. The gene set of interest was obtained from the Molecular Signatures Database (MSigDB) (http://www.broadinstitute.org/gsea/msigdb/) (GO:0002718, GOBP, regulation of cytokine production involved in immune response). Enrichment analysis based on Gene Ontology (GO) terms and KEGG was performed by Enrichr (https://maayanlab.cloud/Enrichr/) (25). Then, figure drawing and graphic display were performed by a website (www.bioinformatics.com.cn), an online platform for data analysis and visualization.