Protoplast Preparation for Algal Single-Cell Omics Sequencing

Abstract

Single-cell sequencing (SCS) is an evolutionary technique for conducting life science research, providing the highest genome-sale throughput and single-cell resolution and unprecedented capabilities in addressing mechanistic and operational questions. Unfortunately, the current SCS pipeline cannot be directly applied to algal research as algal cells have cell walls, which makes RNA extraction hard for the current SCS platforms. Fortunately, effective methods are available for producing algal protoplasts (cells without cell walls), which can be directly fed into current SCS pipelines. In this review, we first summarize the cell wall structure and chemical composition of algal cell walls, particularly in Chlorophyta, then summarize the advances made in preparing algal protoplasts using physical, chemical, and biological methods, followed by specific cases of algal protoplast production in some commonly used eukaryotic algae. This review provides a timely primer to those interested in applying SCS in eukaryotic algal research.

Keywords: Chlorella vulgaris; algal protoplast; bead; cell wall; cellulose; enzymatic lysis; sequencing; single-cell.

Figure 1

Pipeline of algal protoplasts fed into existing single-cell sequencing pipelines.

Figure 2

Cell wall structure and chemical compositions of Haematococcus pluvialis. H. pluvialis cells are morphologically classified into four types: flagellated cells, palmelloid cells, intermediate cells, and cysts. During the cyst period, H. pluvialis’s cell wall has a multilayer structure, including a primary layer on the outside, followed by a trilaminar sheath, a secondary layer, a tertiary wall, and an electron later located between the secondary and tertiary wall. Polysaccharides, proteins, and tough non-hydrolysable sporopollenins are the main constituents of the cell wall.

Figure 3

Cell wall structure and chemical compositions of Chlorella vulgaris. C. vulgaris cell wall has a unilamellar structure, consisting of an inner layer with an electron-dense outer wall and a low-density layer, and an outer layer with hairlike fibers. The cell wall of C. vulgaris is mainly composed of cellulose, hemicellulose, monosaccharides, lipid, protein, and glycosyl. As a glucosamine-type species, glucosamine is the main constituent of the cell wall of C. vulgaris, and proteins constitute about 20% of the cell wall.

Figure 4

General protocols for protoplast isolation from algal cells. The protocol is mainly composed of five steps, including enzyme solution preparation, cell preparation, chelator treatment, enzymolysis, protoplast collection, and protoplast confirmation and viability analysis.