Single-cell RNA-seq analysis of mTECs in Dhx9 cKO mice

Single-cell suspensions of mTEC^hi and mTEC^lo from WT and Dhx9 cKO mice were sorted using a Fusion cell sorter (BD Biosciences), with two independent parallel samples in each group (Figure S2). The operations are consistent with the previously published method (32 (link)). We used DEGseq to identify the differential expressed genes (q-value < 0.05, |log₂FC| > 0) of mTEC^hi and mTEC^lo from the different genotype mice, respectively (33 (link)). According to the differential expressed genes in mTECs of WT and Dhx9 cKO mice, GSVA was performed to calculate the individual gene set enrichment scores, which were visualized by R3.6.0. The analysis of the KEGG pathway was performed by KOBAS 3.0 (34 (link)). GSEA was carried out by searching the KEGG database (34 (link)). All analyses were carried out with P < 0.05 as the cutoff criterion. The raw data of RNA-seq sequencing used in this article could be found in the National Genomics Data Center (NGDC), part of the China National Center for Bioinformation (CNCB) under number PRJCA008466.