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

Sample preparation involved Trizol RNA isolation (∼1 hour) following library preparation, which included the synthesis of dscDNA library for the desired RNA sample (∼2 hours). Samples were barcoded, and adapter ligated (∼1 hour). 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.

List of yeast strains used in this study.

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-hour post-sequencing initiation between HEK293 and HeLa samples. Selected data plots showing sample-to-sample distance plot (B), pPCA (C) and volcano plot (D) 1hr-post sequencing initiation. E-G. Differential gene expression 24-hours 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-hours 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-hours time point), 35 genes were found to be enriched in HEK293 and depleted in HeLa, while 41 genes showed enrichment in HeLa and depletion in HEK293.

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. Gene expression variability. The number of identified genes (> 0 reads counted) is plotted 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 experimental manipulated yeast setup 1 samples using NanopoReaTA.

A. Experimental strategy of yeast setup 1. RNA was isolated from selected yeast strains and dscDNA library was prepared which included samples 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-G. Differential gene expression in WT-pNew1(HIS3) versus WT-pEV(HIS3). Selected data plots showing PCA (B-C) and volcano plots (D-E) 1hr and 24hr post sequencing initiation. F. Normalized gene count for selected genes 24hr 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. H-M. Differential gene expression in new1Δ-pNew1(HIS3) versus new1Δ-pEV(HIS3). Similar analyses to B-G were conducted for PCA (H-I) and volcano plots (J-K) as well as normalized gene counts (L) and Venn diagram (M). N-S. Differential gene expression in new1Δ-pEV(HIS3) versus WT-pEV(HIS3). Similar analyses to B-G were conducted for PCA (N-O) and volcano plots (P-Q) as well as normalized gene counts (R) and Venn diagram (S). T-Y. Differential gene expression in new1Δ-pNew1(HIS3) versus WT-pNew1(HIS3). Similar analyses to B-G were conducted for PCA (T-V) and volcano plots (U-W) as well as normalized gene counts (X) and Venn diagram (Y).

Real-time transcriptomic analysis between experimental manipulated yeast setup 2 samples using NanopoReaTA.

A. Experimental strategy of yeast setup 2. RNA was isolated from selected yeast strains and 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-G. Differential gene expression in rkr1Δ-pJlp2(URA3) versus rkr1Δ-pEV(URA3). Selected data plots showing PCA (B-C) and volcano plots (D-E) 1hr and 24hr post sequencing initiation. F. Normalized gene counts for selected genes 24hr 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. H-M. Differential gene expression in rkr1Δ, jlp2Δ-pJlp2(URA3) versus rkr1Δ, jlp2Δ-pEV(URA3). Similar analyses to B-G were conducted for PCA (H-I) and volcano plots (J-K) as well as normalized gene counts (L) and Venn diagram (M). N-S. Differential gene expression in rkr1Δ-pEV(URA3) versus WT-pEV(HIS3). Similar analyses to B-G were conducted for PCA (N-O) and volcano plots (P-Q) as well as normalized gene counts (R) and Venn diagram (S). T-Y. Differential gene expression in in rkr1Δ, jlp2Δ-pEV(URA3) versus WT-pEV(HIS3). Similar analyses to B-G were conducted for PCA (T-V) and volcano plots (U-W) as well as normalized gene counts (X) and Venn diagram (Y).