Streamlined pipeline for the rapid experimental setup and utilization of NanopoReaTA.

Sample preparation involved Trizol RNA isolation (∼1 hr) following library preparation, which included the synthesis of dscDNA library for the desired RNA sample (∼2 hrs). Samples were barcoded, and adapter ligated (∼1 hr). Sequencing was performed using a PromethION R10 flow-cell or MinION R9 flow cell (for HEK293 and HeLa), and real-time data analysis and visualization occurred alongside ongoing sequencing. For this study, figures were exported at 1hr, 2hr, 5hr, 10hr, and 24hr psi, providing insights into the dynamic transcriptional changes of long-read RNA-seq between distinct conditions.

Real-time transcriptomic analysis between HEK293 and HeLa using NanopoReaTA.

A. Experimental strategy. RNA was isolated from HEK293 and HeLa cells, the dscDNA library was prepared which included sample barcoding and adapter ligation. Samples were loaded and sequenced using a PromethION R10 flow cell. NanopoReaTA was activated shortly after sequencing initiation and data was collected 1hr, 2hr, 5hr, 10hr, and 24hr post-sequencing initiation. B-D Differential gene expression 1-hr post-sequencing initiation between HEK293 and HeLa samples. Selected data plots showing sample-to-sample distance plot (B), Principal component analysis (PCA) (C) and volcano plot (D) 1hr-post sequencing initiation. E-G. Differential gene expression 24-hrs post-sequencing initiation between HEK293 and HeLa samples. Selected data plots showing sample-to-sample distance plot (E), Principal component analysis (PCA) (F) and volcano plot (G) 24-hrs post-sequencing initiation. H. Five-way Venn diagram showing the differentially expressed gene overlaps between the distinct collected time points. I. Validation of identified differentially expressed genes through Harmonizome database (Rouillard et al. 2016). For this analysis, we utilized "HPA Cell Line Gene Expression Profiles" (Uhlén et al. 2015). Among the DEGs (identified at the 24-hrs time point), 19 genes were found to be enriched in HEK293 and depleted in HeLa, while 46 genes showed enrichment in HeLa and depletion in HEK293. One gene (PFN1) was enriched in HeLa and enriched in HEK293 Harmonizome database.

Real-time transcriptomic analysis between rRNA-depleted and rRNA-enriched transcripts using NanopoReaTA.

A. Experimental strategy. RNA was isolated from HEK293 and selective purification of distinct RNA populations was performed using the Ribominus™ Eukaryote kit. The dscDNA library was prepared, which included sample barcoding and adapter ligation. Samples were loaded and sequenced using a PromethION R10 flow cell. NanopoReaTA was activated shortly after sequencing initiation and data was collected 1hr, 2hr, 5hr, 10hr, and 24hr post- sequencing initiation. B-H. Differential gene expression between RiboM and TotalR from HEK293. Selected data plots showing PCA and volcano plots 1hr (B-C) and 24hr (D-E) post sequencing initiation. F. Normalized gene counts for selected genes 24hr. Normalized gene counts are visualized for selected genes per condition using boxplots. The median- of-ratios normalization method from DESeq2 was used for normalization. G. Number of identified genes with >0 aligned reads after each iteration per condition. H. Five-way Venn diagram showing the differentially expressed gene overlaps between the distinct collected time points. I-O. Differential gene expression between RiboP and TotalR. Similar analyses to B-H were conducted for comparisons at 1hr (I-J) and 24hr (K-L) as well as normalized gene counts (M), gene expression variability (N) and Venn diagram (O). P-U. Differential gene expression compared between RiboM and RiboP. Similar analyses to B-H were conducted for comparisons at 1hr (P-Q) and 24hr (R-S) as well as normalized gene counts (T), gene expression variability (V) and Venn diagram (U).

Real-time transcriptomic analysis between heat-shock and non-heat shock treated cells using NanopoReaTA.

A. Experimental strategy. RNA was isolated from HEK293 treated with heat shock on non-heat shock treatment (n=6) and selective purification of distinct RNA populations was performed using the Ribominus™ Eukaryote kit. The dscDNA library was prepared, which included sample barcoding and adapter ligation. Samples were loaded and sequenced using a PromethION R10 flow cell. NanopoReaTA was activated shortly after sequencing initiation and data was collected 1hr, 2hr, 5hr, 10hr, and 24hr post-sequencing initiation. B-D Differential gene expression 1-hr post-sequencing initiation between HEK293 and HeLa samples. Selected data plots showing PCA (B), volcano plot (C) and normalized counts for selected genes (D) 1hr post sequencing initiation. E-G. Differential gene expression 24-hrs post-sequencing initiation between HEK293 and HeLa samples. Selected data plots showing PCA (E), volcano plot (F) and normalized counts for selected genes (G) 24-hrs post-sequencing initiation. H. Five-way Venn diagram showing the differentially expressed gene overlaps between the distinct collected time points. I. Gene Ontology (GO) enrichment analysis of upregulated gene in HS compared to NHs conditions. J. Volcano plot depicting differential transcript usage between HS and NHS conditions.

Real-time transcriptomic analysis between new1Δ-pEV(HIS3) vs WT-pEV(HIS3) using NanopoReaTA.

A. Experimental strategy. Following library preparation (n=3), samples were loaded and sequenced using a PromethION R10 flow cell. NanopoReaTA was activated shortly after sequencing initiation and data were collected 1h, 2h, 5h, 10h, and 24h post-sequencing initiation. B-C. PCA plot showing the sample separation between new1Δ-pEV(HIS3) vs WT-pEV(HIS3) 1- h post-sequencing (B) and 24-h post-sequencing (C). D-E. Volcano plots showing the differentially expressed genes between new1Δ-pEV(HIS3) vs WT-pEV(HIS3) 1-h post-sequencing initiation (D) and 24-h post-sequencing initiation (E). F. Normalized gene count for selected genes 24h post sequencing initiation. Normalized gene counts are visualized for selected genes per condition using boxplots. The median-of-ratios normalization method from DESeq2 was used for normalization. G. Five-way Venn diagram showing the differentially expressed gene overlaps between the distinct collected time points.

Real-time transcriptomic analysis between rkr1Δ-pEV(URA3) vs WT-pEV(HIS3) using NanopoReaTA.

A. Experimental strategy. Following library preparation (n=3), samples were loaded and sequenced using a PromethION R10 flow cell. NanopoReaTA was activated shortly after sequencing initiation and data was collected 1h, 2h, 5h, 10h, and 24h post-sequencing initiation. B-C. PCA plot showing the sample separation between rkr1Δ-pEV(URA3) vs WT-pEV(HIS3) 1- h post-sequencing initiation (B) and 24-h post-sequencing initiation (C). D-E. Volcano plots showing the differentially expressed genes between rkr1Δ-pEV(URA3) vs WT-pEV(HIS3) 1-h post-sequencing initiation (D) and 24-h post- sequencing initiation (E). F. Normalized gene count for selected genes 24h post sequencing initiation. Normalized gene counts are visualized for selected genes per condition using boxplots. The median-of-ratios normalization method from DESeq2 was used for normalization. G. Five-way Venn diagram showing the differentially expressed gene overlaps between the distinct collected time points.

List of yeast strains used in this study.