Deciphering the RNA landscape by RNAome sequencing

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods.

当前的RNA表达谱分析方法需针对特定RNA类别开展富集操作，因此无法以未受扰动的方式检出全部RNA物种。本研究报道了一种链特异性RNAome测序技术，可通过单次测序反应，从核糖体RNA（rRNA）去除的总RNA样本中同时测定小RNA与大RNA的表达水平。鉴于当前分析流程无法可靠地同时分析小RNA与大RNA，本研究开发了TRAP（总RNA分析流程，Total RNA Analysis Pipeline）——一款兼容现有RNA测序方案的稳健分析界面。该RNAome测序技术可在定量层面完整保留所有RNA类别的表达信息，支持跨类别比较，从而助力不同RNA类群间关联关系的鉴定。我们以经顺铂处理的小鼠胚胎干细胞为模型，验证了RNAome测序技术的应用优势。RNAome测序得到的微小RNA（microRNA，miRNA）与信使RNA（messenger RNA，mRNA）表达在生物学重复间显著相关，且与现有RNA测序方法及同批样本的基因表达芯片结果高度一致。此外，RNAome测序还可检出其他方法无法检测到的多种RNA类别，包括增强子RNA、反义RNA、新型RNA物种以及大量差异表达RNA。在整体RNA类别层面，RNAome测序还发现顺铂处理后微小RNA（miRNA）及其异构体呈现全局性特异性表达抑制，而mRNA等其他RNA类别则无明显变化。RNAome测序的这些特性，将显著推动表达分析研究以及现有方法尚未覆盖的RNA生物学研究。