How does 10x chromium work?

10x Chromium is a machine, or platform, that researchers use for high-throughput single-cell RNA sequencing (scRNA-seq). It enables the analysis of gene expression profiles at single-cell resolution. Also, it can operate at a scale of hundreds to ten thousands of cells per run. 10x Chromium is compatible with the droplet-based scRNA-seq methods of 10x Genomics.

Read on to learn about the various Chromium series, how the platform works, and what its features are.

The difference between 10x Chromium X, iX and Controller.

Chromium X Series

The most advanced generation of 10x Chromium instruments is the Chromium X Series. It offers the highest throughput (> 100,000 cells and more) per run and is compatible with the newest single-cell assays.

10x genomics chromium controller

In comparison: the previous 10x Genomics instrument, the Chromium Controller, can run low-throughput (100–1000 cells) and moderate-throughput (1,000–10,000 cells) assays.

10x Genomics Chromium iX

The 10x Genomics Chromium iX is a "light" version that is more cost-effective and has lower throughput in the same machine. It is upgradable to 'full-range' 10x Chromium X with a software update.

How 10x Chromium works

10x Chromium works with microfluidics. By using tiny vessel-like tubes, the system can partition individual cells or nuclei into micro-scale water droplets. These function as tiny reaction chambers in which the RNA molecules from each cell receive barcoded labels and prepare for sequencing.

The 10x Chromium system works with microfluidic chips. Each chip can hold multiple samples simultaneously. With samples, what is meant is a single-cell suspension. Hence, before sequencing can take place, you must dissociate each sample into single-cell suspensions by a tissue-specific protocol.

After loading, this is what happens inside the 10x Chromium System:

Partition into droplets

The system divides individual cells into tiny water droplets and adds reagents and barcoded gel beads. These gel beads are coated with oligonucleotides, which are short nucleotide sequences.

Barcoding

Each oligo comprises two barcoding components and one RNA-binding component. This RNA-binding component is a poly(T)-stretch that attaches to the mRNA.

One of the barcoding components is a unique cellular barcode for each bead. This is linked to each mRNA, allowing it to be traced back to its originating cell. Another section of the oligo uniquely barcodes each RNA molecule. This so-called unique molecular identifier (UMI) has a crucial role in accurately measuring gene expression.

The droplets, known as gel beads in emulsion (GEMs), should each contain a single cell, a barcoded gel bead, and reagents. These reagents include enzymes such as reverse transcriptase and polymerase, and nucleotides. In each GEM, the reagents trigger the cells to release the RNA. Now, the mRNA molecules bind with the barcoded gel beads.