RNA degradation is a concern in paraffin-embedded samples, as well as in samples subjected to repeated freeze-thaw cycles or those that are not promptly and properly preserved. In conventional mRNA degradation senarios, when the poly(A) tail becomes shortened (i.e., deadenylation), the poly(A)-binding protein PABPC dissociates from the mRNA molecule, leading to its subsequent degradation. Traditional single-cell technologies rely on poly(A) for RNA capture, thus requiring high RNA integrity. This results in subpar performance for paraffin-embedded or degraded samples in traditional single-cell methods.

In contrast, bacterial RNA itself lacks a poly(A) structure, rendering other current single-cell technologies incapable of detecting bacterial RNA.

Based on random primer principle, M20 Seq technology can capture RNA molecules and perform single-cell barcoding, even if RNA molecules are partially degraded or lack of a poly(A) structure. Therefore, it can be used for single-cell detection of paraffin-embedded samples, RNA-degraded samples, as well as bacterial samples.

The entire experimental process takes about 1.5-2.5 days from sample dissociation to library construction.

There are several checkpoints during the experiment: post nucleus extraction, post reverse transcription, post cDNA extension, and post cDNA amplification. Processes can be paused at the checkpoints.

To achieve in situ reverse transcription, “M20 Seq”requires effective fixation of samples. 4% paraformaldehyde fixation can terminate any ongoing biochemical reactions, preserve the inherent substances in cells, coagulate intracellular proteins, minimize or terminate the reactions of endogenous and exogenous enzymes, and make cells or tissues basically Remain consistent with the substance in the active state.

Formaldehyde can combine with amino groups and other groups of proteins. When formaldehyde combines with two molecules to make them close together, they will cross-link to form "-CH2-", which is called methylene bridge. The number of crosslinks increases with time, forming a spatial network. Substances such as carbohydrates, lipids, and nucleic acids are trapped in a network of cross-linked proteins. Nucleic acid material does not overflow the nucleus.

(Click to view the fixing principle)

After reverse transcription, a capture adapter is added to the end of cDNA. The capture adapter hybridizes with the oligo sequence on barcoded microbeads. Subsequent extension adds a cell label to the cDNA chain, completing single-cell barcoding.

The performance of the equipment is as follows:
Cell throughput range: 1000-20000 cells/channel (with standard reagent kit);
Sample throughput: 4 channels;
Cell size: no limitations, both cells and cell nuclei are acceptable;
Cell capture rate: 50%~60%;
Doublet rate: less than 2% per 10000 cells;
Processing time per run: less than 15 minutes;
Applicable sample types: fresh, frozen, FFPE (Formalin-Fixed Paraffin-Embedded);
Applicable species: eukaryotes (human, mouse, rat, plants, etc.), prokaryotes (bacteria, etc.).