Preparation of a Single Cell Suspension from the Murine Iridocorneal Angle

Benjamin R. Thomson; Susan E. Quaggin

doi:10.21769/BioProtoc.4426

Preparation of a Single Cell Suspension from the Murine Iridocorneal Angle

Benjamin R. Thomson^*, Susan E. Quaggin

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

Abstract

Single cell RNA sequencing is a powerful tool that can be used to identify distinct cell types and transcriptomic differences within complex tissues. It has proven to be especially useful in tissues of the eye, where investigators have identified novel cell types within the retina, anterior chamber, and iridocorneal angle and explored transcriptomic contribution to disease phenotypes in age-related macular degeneration. However, to obtain high quality results, the technique requires isolation of healthy single cells from the tissue of interest, seeking complete tissue digestion while minimizing stress and transcriptomic changes in the isolated cells prior to library preparation. Here, we present a protocol developed in our laboratory for isolation of live single cells from the murine iridocorneal angle, which includes Schlemm’s canal and the trabecular meshwork, suitable for single cell RNA sequencing, flow cytometry, or other downstream analysis.

Original language	English (US)
Article number	e4426
Journal	Bio-protocol
Volume	12
Issue number	10
DOIs	https://doi.org/10.21769/BioProtoc.4426
State	Published - May 20 2022

Funding

We are grateful to Drs. Robert Lavker and Nihal Kaplan (Northwestern University Feinberg School of Medicine) for helpful discussions and for sharing their protocol for cell dissociation (described in Kaplan et al., 2019), upon which this protocol was based. Automated cell counting and quality control was performed by Dr. Jennifer Chin Man Wai of the Center for Genetic Medicine of the Feinberg School of Medicine. Work described here was funded by R01 EY025799 (to SEQ.), R01 EY032609 (to BRT) and a Brightfocus foundation new investigator grant in macular degeneration research to BRT. Benjamin Thomson receives research funding from Bayer. Susan Quaggin owns stock in Mannin Research Inc. and receives consulting fees from AstraZeneca, Janssen, the Lowy Medical Research Foundation, Novartis, Pfizer, Janssen, and Roche/Genentech.

Keywords

Dissociation
Eye
Glaucoma
Iridocorneal angle
Limbus
Mouse
Schlemm’s canal
Single cell
Single cell RNA sequencing

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology
General Immunology and Microbiology
General Neuroscience
Plant Science

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10.21769/BioProtoc.4426

Cite this

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title = "Preparation of a Single Cell Suspension from the Murine Iridocorneal Angle",

abstract = "Single cell RNA sequencing is a powerful tool that can be used to identify distinct cell types and transcriptomic differences within complex tissues. It has proven to be especially useful in tissues of the eye, where investigators have identified novel cell types within the retina, anterior chamber, and iridocorneal angle and explored transcriptomic contribution to disease phenotypes in age-related macular degeneration. However, to obtain high quality results, the technique requires isolation of healthy single cells from the tissue of interest, seeking complete tissue digestion while minimizing stress and transcriptomic changes in the isolated cells prior to library preparation. Here, we present a protocol developed in our laboratory for isolation of live single cells from the murine iridocorneal angle, which includes Schlemm{\textquoteright}s canal and the trabecular meshwork, suitable for single cell RNA sequencing, flow cytometry, or other downstream analysis.",

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note = "Funding Information: We are grateful to Drs. Robert Lavker and Nihal Kaplan (Northwestern University Feinberg School of Medicine) for helpful discussions and for sharing their protocol for cell dissociation (described in Kaplan et al., 2019), upon which this protocol was based. Automated cell counting and quality control was performed by Dr. Jennifer Chin Man Wai of the Center for Genetic Medicine of the Feinberg School of Medicine. Work described here was funded by R01 EY025799 (to SEQ.), R01 EY032609 (to BRT) and a Brightfocus foundation new investigator grant in macular degeneration research to BRT. Funding Information: Benjamin Thomson receives research funding from Bayer. Susan Quaggin owns stock in Mannin Research Inc. and receives consulting fees from AstraZeneca, Janssen, the Lowy Medical Research Foundation, Novartis, Pfizer, Janssen, and Roche/Genentech. Publisher Copyright: {\textcopyright} 2022 The Authors; exclusive licensee Bio-protocol LLC.",

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Preparation of a Single Cell Suspension from the Murine Iridocorneal Angle

Abstract

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Keywords

ASJC Scopus subject areas

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