important research field. Particularly, the access to cells in situ is of
interest [22]. In combination with immune histological staining,
LCM is a powerful tool for solid sample analysis on the single-cell
level [23]. In the past years, various applications in single-cell
analysis based on LCM-extracted cells have been published:
single-cell RT-PCR [24], short tandem repeat (STR) analysis in
forensics [25], Western blot, and mass spectrophotometry [26].1.1.5 Limiting Dilution Today many laboratories and companies use hand pipettes or pipet-
ting robots to isolate individual cells through dilution of the cell
suspension (Fig.2E). Due to the statistical distribution of the cells
in the suspension, the number of cells in a highly diluted sample can
be as low as one single cell per aliquot, when the suspension is split
into small volumes (aliquots). This process is termed limiting dilu-
tion and is well known for decades for the production of monoclo-
nal cell cultures [27]. Besides antibody production (as done by
hybridomas), other applications such as cell-based assays, etc. also
require cell populations grown from a single cell.
1.2 Sequencing
Transcriptomes of
Single Cells
RNA sequencing (RNA-seq) enabled transcriptomic profiling at
unprecedented sensitivity and breadth, leading to the discovery of
new RNA species and deepening our understanding of transcrip-
tome dynamics [28]. In recent years, low-input RNA-seq methods
have been adapted to work in single cells [29], which introduced a
derived technology called single-cell RNA sequencing (scRNA-
seq). scRNA-seq can quantify intrapopulation heterogeneity and
enable study of cell states and transitions at very high resolution,
potentially revealing cell subtypes or gene expression dynamics that
are masked in bulk, population-averaged measurements
[30, 31]. Over the past years, numerous scRNA-seq protocols
have been developed [29, 32–45], including the widely used
Smart-seq2 [37] and CEL-Seq [35]. Currently published scRNA-
seq protocols all follow the same general workflow: single cells are
isolated (seeformer section); cells are lysed, and the RNA is cap-
tured for reverse transcription into cDNA; and the cDNA is pre-
amplified and then used to prepare libraries for sequencing and
downstream analysis. Since the technology and protocol of each
scRNA-seq approach are out of the scope of this section, readers
refer to [46] for a comprehensive review of individual scRNA-seq
protocols and their relative strengths and weaknesses. Moreover,
two recent studies from the Enard group [47] and the Teichmann
group [48] have compared tens of different protocols, which will
give the readers more comprehensive insights into the scRNA-seq
technologies.Applications of Single-Cell Sequencing for Multiomics 331