Long-read transcriptome profiling of the rat hippocampal neurons in primary culture at different time points after activation using Nanopore sequencing

Long-read transcriptome sequencing provides us with a convenient tool for thorough study of biological processes such as neuronal plasticity. In this study, we aimed to perform transcriptional profiling of rat hippocampal primary neuron cultures after stimulation with picrotoxin (PTX) to further understand molecular mechanisms of neuronal activation. To overcome limitations of short-read RNA-Seq approaches we performed a Oxford Nanopore Technologies (ONT)-based long-read sequencing and transcriptome assembly of rat primary hippocampal culture mRNA at three time points after the PTX activation (30 min; 1 h and 5 h). Overall, we found 23652 novel transcrits in comparison to reference annotations, out of which ~6k were entirely novel and mostly transposable element-derived loci. Analysis of differentially expressed genes (DEG) showed that a total of 3046 genes were differentially expressed, of which 2037 were upregulated and 1009 were downregulated at 30 min after the PTX application, with only 446 and 13 genes differentially expressed at 1 h and 5 h time points respectively. Most notably, multiple genes encoding ribosomal proteins, with high basal expression level, were downregulated after 30 min incubation with PTX; we suggest that this indicates redistribution of transcriptional resources towards activity-induced genes. Novel loci and isoforms, observed in this study, could help us further understand the functional mRNA repertoire in neuronal plasticity processes. Together with other NGS techniques, the result of differential gene expression analysis of sequencing data obtained using MinION platform might provide a simple method to optimize further study of neuronal plasticity. Overall design: 20 primary hippocampal neuronal cultures under control and PTX-treated conditions were subjected to long-read Oxford Nanopore RNA sequencing and transcriptome assembly. 12 cultures (with noticeable response to PTX) were subjected to differential expression analysis.

长读长转录组测序为我们深入研究神经元可塑性等生物学过程提供了高效便捷的研究工具。本研究旨在对经印防己毒素（picrotoxin, PTX）刺激后的大鼠海马原代神经元培养物开展转录组表达谱分析，以进一步阐明神经元激活的分子机制。为克服短读长RNA测序（RNA-Seq）技术的局限性，我们基于牛津纳米孔科技公司（Oxford Nanopore Technologies, ONT）的测序平台，在PTX激活后的三个时间点（30分钟、1小时及5小时）对大鼠海马原代培养物的mRNA进行了长读长测序与转录组组装。总体而言，相较于参考注释序列，本研究共鉴定出23652个全新转录本，其中约6000个为完全新型的转录本，且大多源自转座因子位点。差异表达基因（differentially expressed genes, DEG）分析结果显示，共有3046个基因呈现差异表达特征：在PTX处理30分钟时，分别有2037个基因上调、1009个基因下调；而在1小时和5小时时间点，差异表达基因的数量仅分别为446个和13个。尤为值得关注的是，多个编码核糖体蛋白且基础表达水平较高的基因在PTX孵育30分钟后出现表达下调现象；我们推测该现象表明转录资源发生了重新分配，转向活性诱导基因。本研究中发现的新型基因座与转录本异构体，有助于我们进一步阐明神经元可塑性过程中功能性mRNA的全部组成。结合其他下一代测序（next-generation sequencing, NGS）技术，基于MinION测序平台获得的测序数据所开展的差异基因表达分析结果，或可为神经元可塑性的后续研究提供一种简便的优化方案。实验整体设计如下：共设置20组大鼠海马原代神经元培养物，分别置于正常对照与PTX处理条件下，均进行长读长牛津纳米孔RNA测序及转录组组装；其中12组对PTX处理呈现显著响应的培养物，被用于差异表达分析。