We would like to do single cell RNA-seq to study tumor and its microenvironment (especially immune cells) with or without drug treatment in the mouse model. I found that, in some articles, CD45+ and CD45- cells were separated, and scientists will prepare cDNA library for CD45+ cells and CD45- cell for single cell RNA-seq. However, some scientist directly dissociate tumor and prepare the library, then analyze the single cell RNA-seq. Could someone help me understand the pros and cons of these different approach? Thank you very much!
Those who directly prepare cDNA library from cells, they do not go thru separation step of flow cytometry, probably that step would change the microenvironment of tumor for an hour or so and hence their gene expression is likely to alter. They were able to analyze later their results.
At one place, I had to take this decision, I avoided flow cytometry step. Every one has their own choice.
Good luck
Hello,
If you're interested in the tumour microenvironment (TME) you should be looking at many different cell types. CD45+/- can be useful to have a enrichment in the immune infiltrate, that's correct but then you may deplete many other TME populations that you want to address. The approach that many researchers are following nowadays is to get rid of the tumour cells, because they can use markers as endogenous fluorescence to distinguish them. From some tumours, tumour cells can mean a very high % of the sample, by removing them you're increasing the resolution of your analysis and you could find out more populations within your TME.
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