Whole genome transcriptome of in vivo Treg cells and ex vivo TGF-beta induced Treg cells from WT and Aim2 knockout mice

To understand the whole genome transcriptome of in vivo Treg cells and ex vivo TGF-beta induced Treg cells from WT and Aim2 knockout mice, the total RNA was extracted from indicated Treg cells using the Direct-zol miniprep kit (Zymo Research, R2060). The RNA samples were firstly enriched by Oligo(dT) magnetic beads and used to construct BGISEQ-500 libraries. RNA-seq libraries sequenced using the 50bp single-end protocol (in vivo isolated Treg cells) or 100bp paired-end protocol (TGF-β induced Treg cells) via the BGISEQ-500 sequencer per the manufacturer’s protocol. After filtering of adaptors and low quality reads, clean reads (>26 Million reads per sample for in vivo isolated Treg cells and >40 Million reads per sample for TGF-β induced Treg cells) are mapped to mouse reference genome using HISAT /Bowtie2 tool. Mapping results are stored in BAM files using SAMtools. Total read counts in gene level were summarized using featureCounts function in the Rsubread in R environment, with the R package biomaRt for gene and transcripts mapping. The differential expression (DE) genes were analyzed by DESeq2 package with default setting using total read counts as input, and the adjusted p value (padj) less than 0.05. Examination of differential gene expression of in vivo islolated Treg cells with or without 24-hour TCR stimulation and TGF-beta induced Treg (iTreg) cells at day one from naive CD4 T cells in WT and Aim2 knockout mice.