“…After filter sterilization, the enzymatic solution was then mixed with alga and incubated at 20 °C with shaking at 70 rpm in the dark. Protoplast generation was assessed by M2R staining under the fluorescence microscope [ 9 ].…”
Section: Methods For Preparing Algal Protoplastsmentioning
confidence: 99%
“…Algal protoplasts are cells from which the cell wall is removed, which can then be processed like animal cells for SCS ( Figure 1 ). Fortunately, the methods for generating protoplasts for all major algal phyla have been readily available, as protoplast production and fusion are the starting materials for traditional genetic engineering and state-of-art genome editing with CRISPR ( Clustered Regularly Interspaced Short Palindromic Repeats ) [ 9 , 10 , 11 , 12 ].…”
Section: Introductionmentioning
confidence: 99%
“…For example, Rhodophyta only has the classes Bangiophyceae [ 13 , 14 , 15 ], Florideophyceae [ 13 ], and Porphyridiophyceae [ 14 ]. Most studied brown algae are from the same class Phaeophyceae, e.g., Dictyopteris pacifica, Scytosiphon lomentaria [ 15 ], Petalonia fascia [ 9 ], and Undaria pinnatifida [ 16 ]. In contrast, there are more green algae that have been studied recently, such as Chlorella protothecoides [ 17 ], Ulva prolifera [ 18 ], Haematococcus pluvialis [ 19 ], and Penium margaritaceum [ 20 ].…”
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.
“…After filter sterilization, the enzymatic solution was then mixed with alga and incubated at 20 °C with shaking at 70 rpm in the dark. Protoplast generation was assessed by M2R staining under the fluorescence microscope [ 9 ].…”
Section: Methods For Preparing Algal Protoplastsmentioning
confidence: 99%
“…Algal protoplasts are cells from which the cell wall is removed, which can then be processed like animal cells for SCS ( Figure 1 ). Fortunately, the methods for generating protoplasts for all major algal phyla have been readily available, as protoplast production and fusion are the starting materials for traditional genetic engineering and state-of-art genome editing with CRISPR ( Clustered Regularly Interspaced Short Palindromic Repeats ) [ 9 , 10 , 11 , 12 ].…”
Section: Introductionmentioning
confidence: 99%
“…For example, Rhodophyta only has the classes Bangiophyceae [ 13 , 14 , 15 ], Florideophyceae [ 13 ], and Porphyridiophyceae [ 14 ]. Most studied brown algae are from the same class Phaeophyceae, e.g., Dictyopteris pacifica, Scytosiphon lomentaria [ 15 ], Petalonia fascia [ 9 ], and Undaria pinnatifida [ 16 ]. In contrast, there are more green algae that have been studied recently, such as Chlorella protothecoides [ 17 ], Ulva prolifera [ 18 ], Haematococcus pluvialis [ 19 ], and Penium margaritaceum [ 20 ].…”
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.
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