High-resolution profiling of total RNA in single cells reveals differentiation trajectories of primary human fallopian tube epithelium
Benjamin K Johnson1, Mary Rhodes1, Rebecca A Siwicki2, Marc Wegener2, Ayush Semwal1, H. Josh Jang1, Jacob Morrison1, Pamela Himadewi1, Kelly Foy1, Joshua Schipper4, Larissa L Rossell3, Emily J Siegwald3, Dave W Chesla3, Jose M Teixeira5, Rachael TC Sheridan4, Ting Wang6, Marie Adams2, Timothy J Triche Jr1, Hui Shen1.
1Epigenetics, Van Andel Institute, Grand Rapids, MI, United States; 2Genomics Core Facility, Van Andel Institute, Grand Rapids, MI, United States; 3Corewell Health Accelerator of Research Excellence, Corewell Health System, Grand Rapids, MI, United States; 4Flow Cytometry Core Facility, Van Andel Institute, Grand Rapids, MI, United States; 5Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI, United States; 6Genetics, Washington University School of Medicine, St. Louis, MO, United States
We present Single-cell TOtal RNA Miniaturized sequencing (STORM-seq), a unique molecular identifier (UMI)-containing, full-length single-cell ribo-reduced RNA sequencing protocol, optimized to profile thousands of cells per run. Using 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 and unbiased transposable element expression at the single-cell level. Incorporation of ERCC spike-ins facilitates proper normalization and cross-experiment comparisons, and preserves biologically meaningful intercellular differences in total RNA abundance. Importantly, STORM-seq is characterized by simple library preparation and bioinformatics procedures, and can be executed using standard laboratory equipment and off-the-shelf reagents, thus rendering it widely accessible. We apply STORM-seq to primary human fallopian tube epithelium (FTE), revealing intermediate/transitional cell states, and a putative progenitor cell population. The results support a trajectory from a multipotent 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. Additionally, we observe coordinated transposable element expression, shaping the ciliated lineage trajectory. 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.
This research is supported by National Cancer Institute grant R37CA230748 to HS, Ovarian Cancer Research Alliance fellowship 891749 to BKJ, philanthropic grants from the Grand Rapids Community Foundation, Folz Family Foundation, and Michelle Marie Lunn Hope Foundation to TJT, and startup funds from the Van Andel Institute to TJT and HS..