Single-cell Total-RNA Profiling Unveils Regulatory Hubs of Transcription Factors

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 only captures mature RNA species to measure gene expressions. Here, we develop a one-step total-RNA chemistry-based scRNA-seq method: snapTotal-seq. We benchmarked 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 identified the critical regulatory hubs mediating the cell cycle dynamics. We also applied snapTotal-seq to profile the oncogene-induced senescence and identified the key regulatory hubs governing the entry of senescence. Furthermore, from the comparative analysis of unspliced RNA and spliced RNA, we identified a significant portion of genes whose expression changes occurred 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. Overall design: We performed scRNA-seq with snapTotal-seq protocol on HEK293T cell line to dissect the gene regulatory network of cell cycle. We also applied snapTotal-seq to investigate the TF regulatory network driving oncogene-induced senescence. We utilized HPNE cells with inducible KRASG12D to profile the gene expression dynamics during oncogene-induced senescence. After the activation of KRASG12D with doxycycline, we collected the cells at day1, day2, day3 and day5, and performed scRNA-seq with snapTotal-seq protocol.