10x Chromium: how does it work and what are its features?

The 10x Chromium X is used for 10x Flex samples

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.

10x Chromium X, iX, and Controller

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.

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.

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 it 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.

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For a visual explanation of 10x Genomics, single nucleus sequencing, 10x Flex and more, you can find our information guide.

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Pooling

Following this, the droplets break open and the system pools all GEMs. A multi-step process ensues that eventually produces barcoded duplicates of the mRNA molecules in high amounts. It includes reverse transcription, which copies the RNA code to DNA molecule fragments.

Moreover, it includes amplification with polymerase chain reaction (PCR). This step entails a cycle of duplications so that sufficient RNA exists for proper sequencing.

Every DNA fragment comprises unique barcodes from the gel bead and a portion of the mRNA code. This means that each fragment holds all the necessary information to connect an mRNA molecule to its specific gene and cell.

Sequencing

Reading out the codes of the DNA fragments does not happen inside the 10x Chromium machine. Often, researchers sequence the pooled libraries on next-generation sequencing platforms. At Single Cell Discoveries, we sequence using in-house Illumina NovaSeq X and other sequencers.

After performing next-generation sequencing and data analysis, the result is transcriptomic profiles of the individual cells, used for cell type identification and many other applications.

10x Chromium X workflow
Image by 10x Genomics: the single-cell RNA seq workflow with 10x Chromium.

10x Chromium features

10x Chromium is a black-and-white machine about the size of a microwave. It contains a touch-screen interface to select different single-cell assays.

Beneath the touch screen is a temperature-controlled tray in which you can load a 10x Genomics Chip. Chromium single-cell chips can hold up to 8 or 16 samples. Different single-cell assays require different chips, which have names such as the Next GEM Chip G or Next GEM Chip Q.

10x Genomics has an interactive page that allows you to further explore 10x Chromium X assets.

10x Genomics solutions

At Single Cell Discoveries, we offer multiple 10x Genomics solutions: 10x Genomics Single Cell Gene Expression (3'), Single Cell Gene Expression Flex, Single Cell Immune Profiling (5'), and CITE-seq (which includes cell surface protein data).

Find out everything about our 10x Genomics services in the 10x Information Guide.