1.1.1 Flow Cytometry Among the various types of flow cytometers, the fluorescence-
activated cell sorting (FACS) systems provide the ability to isolate
single cells (Fig.2A). FACS allows phenotypically distinct cells, and
even nuclei, to be sorted into user-defined vessels and lysis buffers,
thus enabling diverse single-cell and single-nucleus protocols to be
applied at significantly higher throughput [6]. Index sorting [10]
additionally allows direct linking of a single cell’s phenotype (e.g.,
surface marker expression, DNA content) with multiomics analysis.
However, large numbers of cells are required as input, and because
the platform currently offers no opportunity to visualize sorted
cells, care must be taken to identify and exclude cell doublets.
1.1.2 Microfluidics Microfluidic technologies, such as Fluidigm C1 [11], have been
widely applied in single-cell omics studies. These technologies
isolate single cells in individual capture sites and initiate nucleic
Fig. 2Schematic overview of single-cell isolation technologies. (A) An example of flow cytometry,
fluorescence-activated cell sorting (FACS). (B) Three microfluidic principles to isolate single cells. (a) An
aqueous stream of cells is broken up into individual droplets in oil containing random distribution of cells;
(b) pneumatic membrane valves use air pressure to close a microfluidic channel by membrane deflection. This
stops the flow and can trap a cell; and (c) hydrodynamic traps are passive elements that only fit single cells
and hold them at one position. (C) The manual isolation using micromanipulator. (D) Schematic view on laser
capture microdissection (LCM) methods. (a) Contact-based via adhesive tapes; (b) cutting with a focused laser
followed by capture with a vessel. Cutout section extracted by gravity and (c) cutting with a focused laser
followed by pressure catapulting with a defocused laser pulse. (E) Isolation of individual cells through dilution
of the cell suspension using hand pipettes or pipetting robots (adapted from [9] and Google image)
Applications of Single-Cell Sequencing for Multiomics 329