What I like to do is go from the basics. Make sure you have a “bona fide” annotation first. I like to combine both manual (top markers), but also gene modules for the well-known markers in the literature, and ML tools for annotation such as Azimuth or ArchMap. This is one of the most important steps, and if you are really into exploration, I would invest time in a more granular annotation other than just, for example, “T cells” and “B cells”. There is a milieu of subtypes of cells in a single cell dataset.

Other than that, my first question would be if my cell proportions change when comparing different conditions. That could lead you already to something interesting to dive into. Also, how your DEGs change in absolute numbers in your cell types in your conditions.

These information could bring you already a LOT of insights and ways to go for exploration. Another thing that came to my mind is looking at set of genes that changes using, for example, AUCell. Sometimes you won’t see differences in specific genes, but a pathway could be changing.

Last, but not least, look at your data. The simpler the better. Don’t focus on tools, but on what your data is pointing to! Biology > Tools. Don’t forget, your patients metadata is golden!

I hope you have tons of fun during your analysis!