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my question is about phagocytosis as response to cancer. It is known that cytotoxic T cell may kill a cancer cell and sends cytokines to phagocytes like macrophage or dendritic cell to engulf and digest the killed cancer cell. From this, my questions:

  1. can one assume that mRNAs of the cancer cells can be captured and sequenced along with those of the phagocyte by scRNAseq? In this case, the phagocyte should show a hybrid expression profile
  2. if 1 holds (that I have observed in practice), can snRNAseq show a similar result? I ask this because in snRNAseq we deal with nuclei not the entire cell. However, I have observed hybrid expression profiles in phagocytes analyzed by snRNASeq.
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    $\begingroup$ It might help if you added links or an explanation of the techniques involved, and what you have found out - particularly about transport of foreign nucleic acids to the nucleus... $\endgroup$
    – bob1
    Commented Jul 20, 2022 at 22:20

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This is a very interesting question and I will give you an outline of the scenario I think is most likely.

I'm using the same hypothesis to explain another common observation in single nuclei RNA-Seq: In all snRNA-Seq data I have analyzed so far I have found reads for mitochondrial genes although they are not transcribed in the nucleus. How did they get there?

When doing single nuclei RNA-Seq the cells necessarily need to be broken to isolate the nuclei. The nuclear envelope is quite stable but most other cellular membranes will be permeabilized. As a consequence, mRNA from all over the cell is liberated and can stick to the nucleus. When the nuclei are isolated, the mRNAs sticking to them will be isolated too and then will be sequenced as part of that nucleus.
In the end it does not matter if the contaminating mRNA is from mitochondria or from cells taken up via phagocytosis.

I have seen mRNA from hemoglobin sticking to cell membranes in single cell RNA-Seq after > 5 washing steps, so I think it is not unlikely to assume mRNA can stick to the nuclear envelope as well.

You could try and see if the non-phagocyte genes have a higher percentage of splicing than the phagocyte genes. Since most mRNA in the nucleus will not be spliced this would be a hint that these genes come from outside the nucleus. How well this works will depend on the technology you are using. 10X only sequences the very 3' end of the mRNAs and you are not guaranteed to have a splice site in there.

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  • $\begingroup$ Thank you for your interesting answer. I use 10x which outputs UMI counts for each gene so I don't have transcript/splicing information. When you say most nuclear mRNAs don't undergo splicing, do you mean splicing happen in the cytoplasm? Otherwise why is it that? It is worth mentioning that in the phagocytes showing such hybrid expression profiles, genes coding MHC II we're highly expressed, meaning they were most probably APCs as well $\endgroup$
    – MCH
    Commented Jul 23, 2022 at 12:48
  • $\begingroup$ Splicing happens in the nucleus but the spliced mRNA is quickly exported to the cytoplasm. This is why is is recommended by 10X to include introns when mapping single nuclei data (page 9 top of right column). Unfortunately, I'm not an expert in immunology and cannot comment on the expression of marker genes in your cells of interest. $\endgroup$
    – PPK
    Commented Jul 25, 2022 at 7:45

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