Put on your dive gear: we're going deep on single-cell and spatial transcriptomics methods!

Single-Cell Methods:

FACS - Fluorescence-activated Cell Sorting isolates cells using fluorescence and laser deflection by staining cells with a dye or by tagging them with antibodies. Capable of sorting >100 cells per experiment. Examples: Smart-Seq, MATQ-Seq, and CEL-Seq

Microdroplets - Single-cells are isolated by flowing cells and reagents through a device to create single-cell containing oil microdroplets. In many cases a bead covered in poly-T sequences is used to capture the 3’-end of RNA transcripts. Cannot recover full-length transcripts. 10,000+ cells at a time, >50% recovery. Examples: 10x Chromium, Complete Genomics DNBelab, and Drop-Seq

Microwells - Instead of isolating cells in droplets, microfluidics or limiting dilution are used to sequester cells into microscopic wells on a plate or a slide. This technique also allows for the sequencing of full-length transcripts. Examples: BD Rhapsody (10,000+ cells), Fluidigm C1 (800+ cells)

Combinatorial Barcoding - The latest advancement in single-cell is the use of combinatorial barcoding to label transcripts within fixed cells without having to use any fancy or expensive instruments up-front. In-cell reverse transcription with a barcoded primer is performed followed by two rounds of splitting the cells into new 96 well plates and ligating new barcodes. A 4th barcoding split is done using a PCR reaction. 10,000+ cells at a time, <50% recovery. Examples: SPLiT-Seq, Parse Biosciences, Scale Biosciences.

Spatial Methods:

Microdissection - Prepared histology slides can be microdissected in two different ways. Traditional laser capture microdissection can be performed and regions sequenced. Alternatively, slides can be labeled with oligo tagged RNAs or antibodies and then those sequence tags released by the exposure of regions of the slide to UV light. Sequencing of the tags tells you which RNAs/Proteins were present in each dissected region. Example: Leica, etc (Laser Capture), Nanostring GeoMx (UV)

Microarray - Histology slides can also be overlaid with RNA capture arrays like the Illumina beadarray to divine spatial information. This was introduced as Slide-seq but has been commercialized by 10x as Visium.

Multiplex FISH - This is basically Fluorescence In-Situ Hybridization on steroids. Histology slides are prepared and exposed to multiple fluorescently tagged RNA probes. Multiple rounds of fluorescent tagging followed by imaging allows for the detection of gene specific signals at the cellular level. Examples: Vizgen/UltiVue MERFISH, Nanostring CosMx

In-Situ Sequencing - Uses sequencing technology to extend random or transcript specific primers in a rolling circle amplification reaction. Sequencing proceeds using 1-2 base labeled probes with imaging after every cycle of probe addition and provides cellular transcript localization. Examples: 10x Xenium, Element Teton