Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

Laboratory of Gene Expression Institute of Biotechnology CAS Biocev Vestec 252 50 Czech Republic

Laboratory of Gene Expression Institute of Biotechnology CAS Biocev Vestec 252 50 Czech Republic Laboratory of Growth Regulators Faculty of Science Palacky University Olomouc 783 71 Czech Republic

Laboratory of Gene Expression Institute of Biotechnology CAS Biocev Vestec 252 50 Czech Republic TATAA Biocenter AB Gothenburg 411 03 Sweden

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