The transcriptome analysis of Wild Type and Tsc1-deficient mTECs by Next Generation Sequencing

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods and to evaluate protocols for optimal high-throughput data analysisMethods:mTECl mRNA profiles of 4 weeks old wild-type (WT) and TSC1 knockout (TSC1-/-) mice were generated by deep sequencing using Illumina HiSeq 3000.Conclusions: Our study revealed that heterozygous deletion of the Tsc1 gene was sufficient to impair the development of mTECs. The defect of mTECs caused by Tsc1 deficiency perturbed thymocyte development and peripheral T cell homeostasis. Tsc1 deficiency did not affect the cell proliferation of mTECs, but increased the apoptosis of mTECs significantly. RNA-seq analysis showed the pathways involved in lysosomal biogenesis, cell metabolism and apoptosis elevated obviously in Tsc1-deficient mTECs compared with wild-type mTECs. Further study showed that Tsc1-deficient mTECs exhibited over-production of ROS and malfunction of lysosome and underwent lysosomal mediated apoptosis. At last, we showed that the impaired development of mTECs could partially reverse by decreasing mTORC1 activity via heterozygous knockout of Raptor.