June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
High-throughput single-cell RNA sequencing of the developing lens
Author Affiliations & Notes
  • Deepti Anand
    Biological Sciences, University of Delaware, Newark, Delaware, United States
  • Atul Kakrana
    Center for Bioinformatics and Computational Biology, University of Delaware, Newark, Delaware, United States
  • Rosanne Skinner
    Department of Anatomy and Cell Biology, University of Kansas Medical Center Bookstore, Kansas City, Kansas, United States
  • Clark Bloomer
    Department of Anatomy and Cell Biology, University of Kansas Medical Center Bookstore, Kansas City, Kansas, United States
  • Irfan Saadi
    Department of Anatomy and Cell Biology, University of Kansas Medical Center Bookstore, Kansas City, Kansas, United States
  • Salil Anil Lachke
    Biological Sciences, University of Delaware, Newark, Delaware, United States
    Center for Bioinformatics and Computational Biology, University of Delaware, Newark, Delaware, United States
  • Footnotes
    Commercial Relationships   Deepti Anand None; Atul Kakrana None; Rosanne Skinner None; Clark Bloomer None; Irfan Saadi None; Salil Lachke None
  • Footnotes
    Support  NIH/NEI R01 EY021505, NIH/NEI R01 EY029770; Knights Templar Eye Foundation Pediatric Ophthalmology Career Starter Award
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 839. doi:
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    • Get Citation

      Deepti Anand, Atul Kakrana, Rosanne Skinner, Clark Bloomer, Irfan Saadi, Salil Anil Lachke; High-throughput single-cell RNA sequencing of the developing lens. Invest. Ophthalmol. Vis. Sci. 2022;63(7):839.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The eye lens contains anteriorly localized epithelial cells (anterior epithelium of lens, AEL) and posteriorly localized fiber cells (FCs). Cells in the AEL are further distinguished based on their proliferative indexes, while FCs can be distinguished based on their differentiation stages. Thus far, lens transcriptomics has focused on whole lens tissue samples or entire populations of AEL or FCs, and has provided key insights into lens pathobiology. Now, with recent advances, single-cell RNA-sequencing (scRNA-seq) can inform on cell-specific transcript heterogeneity and lead to new insights into pathological changes in individual cells that may contribute toward cataract. To make this approach practicable for wide applicability, we developed a working protocol for lens scRNA-seq that can be applied to tissue samples subjected to short-term storage, and used it to characterize embryonic and newborn mouse lens

Methods : Mouse lenses were micro-dissected at embryonic day (E) 16.5 and postnatal day (P) 0 followed by dissociation with Collagenase/Dispase and TrypLE to obtain a single-cell suspension, followed by cell viability/counts estimates. The cell suspension was frozen for transport and thawed and further processed prior to scRNA-seq library preparation and automated sequencing. Data was analyzed using 10x Genomics data-analysis and Seurat tools

Results : We established a protocol to enzymatically dissociate E16.5 and P0 lenses to obtain a viable cell suspension that can be frozen and thawed and yet be amenable for making scRNA-seq libraries that can yield high-quality RNA for RNA-seq. 10x Genomics tools were used to assign unique molecular identifiers to the expressed transcripts and identify established lens markers. We detected 21,031 cells with total 17,689 genes for E16.5 and 14,108 cells with 17,986 genes for P0. Based on expression profiling and clustering analysis, several distinct cell populations could be distinguished. In addition to known AEL and FC genes, several novel genes in these distinct cell populations were identified

Conclusions : This high-throughput approach to study the lens at single-cell resolution suggests that AEL and FCs can be distinguished further into subsets based on distinct expression profiles. Applying this new approach to lens knockout mouse models will help identify disease-specific cell populations and expedite gene discovery, and thus may find wide applicability in lens studies

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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