I recently did a scRNA seq, and when I started to analyze the data, I am confused about the data, hope you can give me some inputs. Thank you in advance!
We used human CD4+ enrichment kit to isolate CD4 cells from PBMCs, then stained the cells with CD4,CD25,CD127, and flow sorted live CD4+CD25+CD127- cells, and directly send the cells to the facility for scRNA seq on the same day.
When the data came back, we found only 18% of the cells have CD4 expression, and 26% of the cells have CD25 expression. In my opinion, the cells should all have CD4, CD25 expression. The core facility staff told us it is not uncommon, but I am confused.
Please let me know your experience.
Thank you a lot!!
Hi Yan. There are actually many reasons why you cannot see CD4 expression on most of your cells:
- Dropouts: This is the most common phenomenon when you look at transcript level of single cells. It is way more common when you use a cost-effective droplet-based method, such as 10x which sensitivity is not as great as others.
- Protein/mRNA differences: sorted methods are based on the (surface) protein expression. Meanwhile, scRNA-Seq can only quantify the expression at transcript level. So there can be a chance where your CD4 cells stop producing CD4 transcript (simply because they have enough of that glycoprotein? - I don't know)
- Dying cells: Have you looked at the mitochrondrial gene ratio? Maybe your cells were stressed and dying. mRNAs are kinda sensitive and will quickly degrade under such circumstances. I would also recommend you to check all the quality control stats of your data (total count per cell/ total expressed gene/etc) before jumping on any specific gene of interest.
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