Next generation sequencing for quantitative analysis of double-negative (DN) thymocyte transcriptomes from Toxf/f and Toxf/fCd4Cre mice.

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the difference of NGS-derived transcriptome profiling between DN thymocytes from Toxf/f and Toxf/fCd4Cre mice. Methods: mRNA profiles of DN thymocytes sorted by FACS from 3-4-week-old Toxf/f and Toxf/fCd4Cre mice were generated by deep sequencing, in triplicate, using Illumina Novaseq6000. The sequence reads that passed quality filters were analyzed at the transcript level with qRT–PCR using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (Build Ensembl_release100) and identified 19,045 transcripts in the DN thymocytes of Toxf/f and Toxf/fCd4Cre mice with BWA workflow. RNA-seq data showed approximately 1.1% of the transcripts which were differentially expressed between the DN thymocytes from Toxf/f and Toxf/fCd4Cre mice, with the parameter of false discovery rate (FDR) < 0.05 and absolute fold change ≥ 2. Conclusions: Our study exhibits the detailed analysis of DN thymocyte transcriptomes with biologic replicates, generated by RNA-seq technology. The optimized data analysis here provides a framework for comparative investigations of expression profiles. The RNA-seq data allow researchers to identify the transcripts affected by TOX and to better understand the role of TOX in the development of thymocytes.