An optimized protocol for retina single-cell RNA sequencing.

Molecular vision (2020-10-23)
Benjamin R Fadl, Seth A Brodie, Michael Malasky, Joseph F Boland, Michael C Kelly, Matthew W Kelley, Erich Boger, Robert Fariss, Anand Swaroop, Laura Campello
ABSTRACT

Single-cell RNA sequencing (scRNA-seq) is a powerful technique used to explore gene expression at the single cell level. However, appropriate preparation of samples is essential to obtain the most information out of this transformative technology. Generating high-quality single-cell suspensions from the retina is critical to preserve the native expression profile that will ensure meaningful transcriptome data analysis. We modified the conditions for rapid and optimal dissociation of retina sample preparation. We also included additional filtering steps in data analysis for retinal scRNA-seq. We report a gentle method for dissociation of the mouse retina that minimizes cell death and preserves cell morphology. This protocol also results in detection of higher transcriptional complexity. In addition, the modified computational pipeline leads to better-quality single-cell RNA-sequencing data in retina samples. We also demonstrate the advantages and limitations of using fresh versus frozen retinas to prepare cell or nuclei suspensions for scRNA-seq. We provide a simple yet robust and reproducible protocol for retinal scRNA-seq analysis, especially for comparative studies.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
D-(+)-Glucose, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.5%
Sigma-Aldrich
Bovine Serum Albumin, fatty acid free, low endotoxin, lyophilized powder, BioReagent, suitable for cell culture, ≥96% (agarose gel electrophoresis)
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Hanks′ Balanced Salt solution, Modified, with sodium bicarbonate, without phenol red, liquid, sterile-filtered, suitable for cell culture
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Catalase from bovine liver, powder, suitable for cell culture, 2,000-5,000 units/mg protein
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(+)-α-Tocopherol acetate, BioReagent, suitable for insect cell culture, ~1360 IU/g
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Antipain dihydrochloride from microbial source, protease inhibitor
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DL-Cysteine, technical grade