Transcriptome in naturally-occuring self-reactive thymocytes

In the Nur77/Nr4a1-eGFP BAC Tg reporter mice, GFP expression positively correlates with TCR signaling strength. We postulate that among post-selection thymocytes, GFP expression reflects self-antigen recognition across the TCR repertoire. Here we identify differentially expressed transcripts in semimature SP4 thymocytes (CD25- CD69+ CD24+) from Nur77-eGFP BAC Tg reporter mice sorted on 10% highest, intermediate, and lowest GFP expression. Examination of semi-mature SP4 thymocytes with low, medium, and high self-reactivity as marked by Nur77-eGFP reporter expression. Thymi were harvested from Nur77-eGFP reporter mice, and cells were stained for live/dead fixable near-IR, CD25, CD24, CD69, CD4, and CD8a. Cells were gated on the basis of live CD25- CD24+ CD69+ CD4+ CD8a-, and then sorted on the basis of GFP expression. ~10% highest, ~10% medium, and ~10% lowest GFP-expressing cells were sorted directly into RLT buffer for generation of RNAsequencing data sets to identify differentially expressed genes. Biological triplicates of each GFP subset was obtained.