I didn't get what you mean by red blood cell lysing, so my suggestion may not be applicable to your protocol. Anyway, I was able to fix a similar issue using Benzonase for the dissociation of neural organoids. You may try adding Benzonase to the dissociation solution instead of using a regular nuclease. In your case, cell death during harsh or prolonged dissociation may cause releasing an excessive amount of nucleic acids to the medium, which leads cells to adhere to each other. I think you can safely add Benzonase into your dissociation solution to separate individual gland cells, which is able to digest free DNA and RNA molecules with no certain buffer, pH, or temperature requirement. Btw, I assume you won't use the cells to prepare a cDNA library right after dissociation as residual nuclease can hurt your sample. Otherwise, be cautious and practice multiple washes to get rid of any leftovers.

Thanks a lot Volkan Ergin !

With regard to red blood cell lysing, I used ACK lysing buffer to lyses the erythrocytes in the cell suspension. After this step and centrifuge, cell clumps appeared.

I noticed that you recommended adding Benzonase to the dissociation solution. Do you think it can be used again to separate aggregated cells if cell clumps appear again during the wash and centrifuge process?

BTW, Benzonase does not affect following single-cell RNA sequencing, right?

Hi Jingliang, you're welcome. I don't know what ACK is, but adding BSA to the resuspension solution may help to keep cells apart following spin. Regarding benzonase, I recommended it as a better replacement, because you mentioned that you are using RNase cocktail. As long as cells are intact, and well washed by spins in a proper solution (w/o benzonase), it should be ok to go. However, I am not 100% confident in saying it doesn't affect your subsequent steps. So, you may want to give it a try by assessing RNA or cDNA integrity before taking a step forward.

Thank you for your detailed answer Volkan Ergin .

I'm trying benzonase now. Once the experiment is finished, I will feed the result back to you.

Jingliang: My lab is facing the similar problem with dissociated tongue epithelial cells. How was the result of your benzonase use? Thanks.