Department of Molecular and Human Genetics, Houston, TX, USA.
Genetics & Genomics Program, Houston, TX, USA.
Nat Commun. 2024 Jul 15;15(1):5941. doi: 10.1038/s41467-024-50291-3.
Recent development of RNA velocity uses master equations to establish the kinetics of the life cycle of RNAs from unspliced RNA to spliced RNA (i.e., mature RNA) to degradation. To feed this kinetic analysis, simultaneous measurement of unspliced RNA and spliced RNA in single cells is greatly desired. However, the majority of single-cell RNA-seq chemistry primarily captures mature RNA species to measure gene expressions. Here, we develop a one-step total-RNA chemistry-based single-cell RNA-seq method: snapTotal-seq. We benchmark this method with multiple single-cell RNA-seq assays in their performance in kinetic analysis of cell cycle by RNA velocity. Next, with LASSO regression between transcription factors, we identify the critical regulatory hubs mediating the cell cycle dynamics. We also apply snapTotal-seq to profile the oncogene-induced senescence and identify the key regulatory hubs governing the entry of senescence. Furthermore, from the comparative analysis of unspliced RNA and spliced RNA, we identify a significant portion of genes whose expression changes occur in spliced RNA but not to the same degree in unspliced RNA, indicating these gene expression changes are mainly controlled by post-transcriptional regulation. Overall, we demonstrate that snapTotal-seq can provide enriched information about gene regulation, especially during the transition between cell states.
近年来，RNA 速度的发展利用主方程来建立从未剪接 RNA 到剪接 RNA（即成熟 RNA）再到降解的 RNA 生命周期的动力学。为了进行这种动力学分析，非常希望能够在单细胞中同时测量未剪接 RNA 和剪接 RNA。然而，大多数单细胞 RNA-seq 化学主要捕获成熟的 RNA 种类来测量基因表达。在这里，我们开发了一种基于一步总 RNA 化学的单细胞 RNA-seq 方法：snapTotal-seq。我们使用多种单细胞 RNA-seq 测定方法对其在 RNA 速度的细胞周期动力学分析中的性能进行了基准测试。接下来，通过转录因子之间的 LASSO 回归，我们确定了介导细胞周期动态的关键调节枢纽。我们还应用 snapTotal-seq 来分析致癌基因诱导的衰老，并确定控制衰老进入的关键调节枢纽。此外，通过未剪接 RNA 和剪接 RNA 的比较分析，我们确定了一部分基因的表达变化仅发生在剪接 RNA 中，而在未剪接 RNA 中没有相同程度的变化，这表明这些基因表达变化主要受转录后调控控制。总的来说，我们证明了 snapTotal-seq 可以提供关于基因调控的丰富信息，特别是在细胞状态转变期间。
