M20 Genomics
  • Q What is the principle of “M20 Seq” technology?
    A

    “M20 Seq”technology is based on the random primer principle. It enables single-cell barcoding and sequencing after in situ reverse transcription within cells (nucleus) or bacteria. This approach allows for unbiased capture of full-length RNA molecules, including non-coding RNAs such as lncRNA and circRNA. With above, M20 Seq technology enables multidimensional research at the single-cell level.

  • Q Why can “M20 Seq” be used for single-cell detection of paraffin-embedded samples, RNA-degraded samples, and bacterial samples?
    A

    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.

    Comparison chart of “M20 Seq” and poly(A)-based single-cell technologies

  • Q What are the components of the VITA single-cell full-length transcriptome platform based on “M20 Seq” technology?
    A

    VITA single-cell full-length transcriptome platform includes VITApilote series of reagent kits, VITAcruizer single-cell partitioning system, and VITAseer software。

  • Q What is the experimental workflow of the VITA platform?
    A

    The experimental workflow is as follows:

    Experimental flow chart

  • Q How long does the entire experimental process take, and which steps can be paused during the experiment?
    A

    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.

  • Q In situ reverse transcription in cells (nuclei)/bacteria, will the nucleic acid material spill out of the cell (nucleus)?
    A

    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)

  • Q How can single-cell barcoding be performed without Poly(A) capture?
    A

    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.

    Single-cell barcoding schematic diagram

  • Q How is the performance of the equipment?
    A

    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.).

  • Q Is the data from VITA platform compatible with other single-cell analysis software?
    A

    Initial data processing requires VITAseer , and subsequent data analysis is compatible with other single-cell analysis software.

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