Single-cell Total RNA Miniaturized sequencing (STORM-seq) reveals differentiation trajectories of primary human fallopian tube epithelium

Abstract

We present Single-cell TOtal RNA Miniaturized sequencing (STORM-seq), a full-length single-cell ribo-reduced RNA sequencing protocol, optimized to profile thousands of cells per run. Using off-the-shelf reagents and random hexamer priming, STORM-seq recovers comprehensive RNA profiles from single cells with library complexity approaching that of bulk RNA-seq. STORM-seq identifies thousands of additional coding and non-coding transcripts not detected by existing methods, and recovers clinically relevant structural variants at the single-cell level. We apply STORM-seq to primary human Fallopian tube epithelium (FTE), a complex solid tissue, key to both human reproductive biology and ovarian carcinogenesis. In differentiation trajectory analyses, the improved resolution from STORM-seq reveals intermediate/transitional cell states, and a putative progenitor cell population. The results support a trajectory from a bipotent progenitor population to ciliated and secretory cell types in normal FTE. These findings are consistent across human subjects, sequencing depths, and platforms. Long intergenic non-coding RNAs (lincRNAs) appear as key driver genes in both ciliated and secretory lineage trajectories, underscoring the importance of both coding and non-coding RNA in this tissue. By capturing essentially complete individual cellular transcriptomes, STORM-seq sheds new light on the transcriptional programs that establish cellular state and fate in complex tissues.

Quantitative clonal analysis reveals vast heterogeneity among fallopian tube cells for ovarian cancer-initiation and progression

“…While cellular heterogeneity among FT Pax8+ cells has been revealed by both our spatial profiling data and recent single cell sequencing data (Dinh et al, 2021;Hu et al, 2020;Johnson et al, 2022;Ulrich et al, 2022), there are very limited overlaps in the putative stem/progenitorlike signatures from these studies, likely due to exceptional heterogeneity among the Pax8 population, technical limitations, or both. For example, our spatial profiling experiment is most likely confounded by the fact that most cells within the large clones have differentiated into secretory or ciliated cells, thus diluting the stem signatures of the clonal founders.…”

Inferring single-cell and spatial microRNA activity from transcriptomics data

“…We were hoping to explore the relationship between expression and activity of miRNAs using additional total-RNA datasets generated by the novel VASA-seq [46] and STORM-seq [47] technologies. However, we found that these technologies report very low expression values of miRNAs, probably due to low miRNA capture efficiency.…”

“…The concept of adding polyA tails to nonpolyadenylated RNA strands, which was first introduced by Turchinovich et al [44] for sequencing of small (< 150 bp) DNAs or RNAs, has later matured into the methods now known as "totalRNA single cell sequencing". This approach detects RNA strands of all lengths by adding polyA tails to nonpolyadenylated RNA, either before or after fragementation [45][46][47].…”