Differential expression profiles of long non-coding RNAs during the mouse pronucleus stage under normal gravity and simulated microgravity

We report the application of single-molecule-based sequencing technology for high-throughput profiling of lncRNA in mouse cells. By obtaining over 47 million bases of sequence, we identified 3,307 lncRNAs based on single-cell RNA sequencing (RNA-Seq) data, discovered 52 lncRNAs that were significantly differentially expressed, and then selected 10 lncRNAs for qRT-PCR validation. Furthermore, we found that 12 lncRNAs with the target gene Tubb4b or Actn4 might be related to the expression of microtubule and microfilament proteins and the binding relationship was confirmed in a dual-luciferase reporter assay. Finally, Gene Ontology (GO) analysis revealed that the target genes of the differentially expressed lncRNAs participated in cellular processes related to protein transport, binding, catalytic activity, membrane-bounded organelles, protein complexes and the cortical cytoskeleton. We believe the paper will be of particular interest to the readers of your journal as lncRNAs regulated the progress of the mouse early embryo development. Overall design: LncRNA profiles of two differently gravity during the mouse pronucleus stage were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500.本研究报道了单分子测序技术在小鼠细胞长链非编码RNA（long non-coding RNA，lncRNA）高通量表达谱分析中的应用。通过获取超4700万碱基的测序数据，我们基于单细胞RNA测序（RNA-Seq）数据鉴定出3307个lncRNA，其中52个呈现显著差异表达，并选取10个lncRNA进行定量实时聚合酶链反应（qRT-PCR）验证。此外，我们发现12个靶向基因Tubb4b或Actn4的lncRNA可能与微管蛋白及微丝蛋白的表达相关，且二者的结合关系通过双荧光素酶报告基因实验得以验证。最后，基因本体（Gene Ontology，GO）富集分析显示，差异表达lncRNA的靶基因参与了蛋白质转运、分子结合、催化活性、膜结合细胞器、蛋白质复合物及皮层细胞骨架相关的细胞生物学过程。鉴于lncRNA可调控小鼠早期胚胎发育进程，我们认为本文对贵刊读者具有重要参考价值。整体实验设计：针对小鼠原核期两个不同重力条件下的样本，采用Illumina HiSeq 2500平台进行深度测序，设置三次生物学重复以获取lncRNA表达谱。