Aire-KO MEChi RNAseq profiling

Background: In order to become functionally competent but harmless mediators of the immune system, T cells undergo a strict educational program in the thymus, where they learn to discriminate between self and non-self. This educational program is, to a large extent, mediated by medullary thymic epithelial cells (mTECs) that have a unique capacity to express, and subsequently present a large fraction of body antigens. While the scope of promiscuously expressed genes by mTECs is well established, relatively little is known about the expression of variants that are generated by co- and post-transcriptional processes. Results: Our study reveals that in comparison to other cell types, mTECs display significantly higher levels of alternative splicing, as well as A-to-I and C-to-U RNA editing, which thereby further expand the diversity of their self-antigen repertoire. Interestingly, Aire, the key mediator of mTECs promiscuous gene expression, plays a limited role in the regulation of these transcriptional processes. Conclusions: Our results highlight RNA processing as another layer by which the immune system assures a comprehensive self-representation in the thymus which is required for the establishment of self-tolerance and prevention of autoimmunity. Identification of the number of genes expressed in Aire-KO MEChi ~100ng of total RNA was isolated by Trizol extraction from MHC-II high mTECs from a pool of 3 Aire-KO mice. Poly-A-selected transcriptome libraries were generated using the non-directionnal TruSeq V3 RNA Sample Prep Kit (without additional pre-amplification) following the manufacturer's protocols. Enrichment of DNA fragment with adapter molecules on both ends was done using 15 cycles of PCR amplification using the Illumina PCR mix and primer cocktail. Paired-end (2 × 100 bp) sequencing was performed using the Illumina HiSeq2000 machine.

背景：为了成为功能成熟且无害的免疫系统介导因子，T细胞在胸腺中经历严格的教育程序，在此过程中学会区分自身与非自身抗原。这一教育程序在很大程度上由髓质胸腺上皮细胞（medullary thymic epithelial cells，mTECs）介导，这类细胞具有独特的能力，可表达并呈递体内绝大多数抗原。尽管mTECs异位表达的基因范围已得到充分阐明，但对于由共转录及转录后过程产生的转录本变体的表达情况，目前所知甚少。
结果：本研究发现，与其他细胞类型相比，mTECs的可变剪接水平以及A-to-I和C-to-U RNA编辑水平均显著更高，从而进一步扩充了其自身抗原库的多样性。值得注意的是，介导mTECs异位基因表达的关键因子Aire，在调控这些转录后过程中仅发挥有限作用。
结论：本研究结果凸显了RNA加工过程是免疫系统确保胸腺内全面呈现自身抗原的另一层调控机制，而这一机制对于建立自身免疫耐受、预防自身免疫疾病至关重要。
实验方法：从3只Aire基因敲除（Aire-KO）小鼠的MHC-II高表达mTECs混合样本中，通过Trizol法提取总RNA，获取约100ng总RNA以鉴定Aire-KO小鼠mTECs的表达基因数量。采用非定向TruSeq V3 RNA样本制备试剂盒（无需额外预扩增），严格遵循制造商说明书构建Poly-A富集的转录组文库。使用Illumina PCR混合液及引物混合物，通过15轮PCR扩增富集两端带有接头分子的DNA片段。采用Illumina HiSeq2000测序平台进行双端（2×100 bp）测序。