We overview single-cell RNA sequencing, at both small and large scales.
Single-cell sequencing is a technology that provides a snapshot of the transcriptome (i.e., the set of gene expressions) of a collection of individual cells. We present a brief ovewview of single-cell sequencing.
The oligo is made of up several smaller parts.
(c) Using a microfluidic device, trap each cell inside a droplet along with a barcoded bead.
(d) Lyse the cells within the droplets.
(e) The mRNAs bind the oligos. Use reverse transcription to generate cDNAs hybridized to the bead surface.
(f) Wash the cDNAs away from the beads, and sequence in bulk (see this post for a review of sequencing).
(g) Use software (e.g., cellranger) to generate the cell-by-gene expression matrix. This roughly involves (i) collapsing all reads with the same UMI into a single read, (ii) determining the cell from which a read came, and (iii) mapping the read onto a reference genome to determine the gene from which the read came.
There is a major limitation to standard (or small-scale) single-cell sequencing: each droplet can contain at most one cell. If multiple cells are captured in a single droplet, then their transcriptomes are impossible to distinguish.
Experimenters do not have exact control over how many cells are captured in a droplet; this quantity is Poisson-distributed. By experimental design, the majority of droplets contain zero cells.
Large-scale single-cell sequencing enables exciting new applications in single-cell CRISPR screens, cell atlases, and population genomics.
sci-RNA-seq3 (a method related to scifi-RNA-seq) was used to generate an atlas of human fetal tissue.