Deciding Between 10x Genomics GEM and Flex Kits
What should we choose?
Picking the right kits is super important for getting good results in our projects. When it comes to mRNA gene expression, we have two main choices from 10x Genomics: their GEM kits and Flex kits. Each kit has its own strong points and works best for different things. Knowing what sets them apart can really help us make smart choices. So, in this blog, I'll explain the main differences between both kit types and tell you what we need to think about when deciding between them.
1. Understanding the kits
Single-cell gene expression GEM Kits
Examples of single cell gene expression GEM kits are the Single Cell Gene Expression kit (3’ GEM kit) and the Single Cell Immune Profiling kit (5’ GEM kit). Both are based on mRNA reverse transcription.
With the 3’ GEM kit, we can use cells or nuclei derived from fresh, cryopreserved, or frozen tissues. In the case of the 5' kit, we will only be able to use cells so frozen tissue is discarded. Upon loading onto the chromium controller, iX or X, the cells or nuclei undergo encapsulation within oil droplets. These oil droplets, known as GEMs (Gel Beads in Emulsion), will serve as micro-PCR tubes.
Within each GEM, a single cell is paired with a unique bead. This bead will be tagged with oligo-barcoded tails. Beside a common cell identifier, each tail will have a unique molecular identifier (UMI) and will end with a sequence of Thymines (polyT). The polyT sequence will facilitate the capture of the polyA mRNA, which will be released upon cell lysis. Subsequently, the mRNA will be converted into complementary DNA (cDNA) through reverse transcription.
Single-cell gene expression Flex Kit
This kit was designed for formalin-fixed cells or nuclei that can be isolated from fresh, cryopreserved, frozen, or formalin-fixed paraffin-embedded tissues (FFPE). It is a probe-based assay and it needs to be used on the chromium iX or X. The off-the-shelf probe panels are designed against most of the mRNA expressed in a cell (the whole transcriptome), excluding genes like ribosomal, HLA (human leukocyte antigen), KIR (Killer cell immunoglobulin-like Receptors) and TCR (T cell receptor). Custom probes can also be added to our experiments.
After we have our cells or nuclei fixed, they will be permeabilized, and probes will be hybridized with the cells or nuclei mRNA. Next, GEMs will be formed as described before. However, here, the bead tails were designed to capture the probes that are hybridized with the mRNA. After ligation and extension, the probed sections of the mRNA will be amplified.
2. What species are compatible with these kits?
The GEM and Flex kits are compatible with human and mouse species. Then the 3’ and 5’ GEM kits can also be used for rats and non-human primates. On top of that, the 3’ GEM kit can also be applied to marine species, plants, and insects.