M20 Genomics
  • Q What advantages does using FFPE samples offer for single-cell transcriptome analysis?
    A

    1. Sample preparation and preservation are more user-friendly.FFPE samples,fixed with formalin and embedded in paraffin, can be stored and transported at room temperature or 4℃ for extended periods.
    2. Utilizing well-preserved FFPE samples in biorepositories enables retrospective studies, markedly reducing sample collection time in research projects. Additionally, it eliminates the uncertainty regarding the inclusion of fresh clinical samples in data analysis.
    3. FFPE samples allow uniform processing and sequencing, reducing batch effects.
    4. FFPE samples suit tissues susceptible to cell fragmentation due to excessive dissociation.

  • Q Does the process of deparaffinization, rehydration, and dissociation of FFPE samples have an impact on the transcriptional state of the cells?
    A

    Deparaffinization and dissociation of FFPE samples do not influence cellular transcriptional state, as the transcriptome state of FFPE samples is already fixed.

  • Q Are there any limitation to FFPE samples, and what are the quality control requirements?
    A

    Due to potentially severe degradation of RNA over prolonged storage, we recommend to use FFPE samples within 3 years. Prior to single-cell experiments, we advise to conduct DV200 quality control to assess the degree of RNA degradation.

    DV200>40%: qualified;
    DV200≤40%: risky;
    DV200≤30%: Not recommended to proceed.

  • Q Is a minimum paraffin section thickness of 25 μm necessary, or are 5-10 μm thickness slides acceptable?
    A

    The diameters of cell nuclei are generally 5-10μm. Slicing to this thickness would result in the fragmentation of many cell nuclei, with cellular organelles or membranes exposed in the suspension. Although fragments can be removed with centrifugation or commercial kits, excessive impurities may still lead to substantial loss of cell nuclei. Moreover, fragments may adhere to cell nuclei and cause contamination. To minimize fragment generation during the initial separation step, a paraffin section thickness of 25 μm or more is required.

  • Q When emplying the random primer principle, how is the issue of ribosomes addressed?
    A

    For single-cell nucleus transcriptome analysis of FFPE samples and frozen tissues, the proportion of rRNA within cell nuclei is relatively low, necessitating no additional handling. However, rRNA depletion procedure is required for single-cell cytoplasm transcriptome detechtion.。

  • Q Do fresh cells also requires fixation?
    A

    Yes, effective fixation is a prerequisite for in situ reverse transcription, helping lock in cellular transcriptome status.

  • Q Are there significant differences in the single-cell transcriptome results between fresh and FFPE samples?
    A

    According to our previously published data, when testing fresh samples and FFPE samples from the same mouse kidney, the results of both sample types show a significant correlation and high consistency(over 90%).

  • Q What is the recommended number of cells and sequencing depth?
    A

    It is recommended to capture 5000-10000 cells and conduct sequencing with a depth of 100-150 billion bases (100-150G) or 300-450M reads.

  • Q A transcript may combine multiple random primers to produce multiple UMIs, how is the expression normalized?
    A

    Normalization is performed using the SCTransform algorithm. Based on R language, objects of seurat are constructed first, cells and genes are quality controlled and filtered, and then SCTransform is performed.

  • Q What are the proportions of lncRNA and mRNA content in the results?
    A

    Proportions may vary across different tissue type. Generally, lncRNA counstitutes approximately 10%, while mRNA makes up around 80%.

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