Dear all,

I am analyzing my bulk RNA sequencing data now and I would like to conduct a GSEA analysis based on the counts file.

Because of the lack of proper prior data set in the MSigDb, I have to make a prior data set by myself. I read some literature and see how they make it. For example, in the figure4c of paper from Mijnheer,G et al, they use the core Treg genes as prior gene set. I traced back to the literature which they used for setting the prior gene set. The core Treg gene list actually includes the down- and up- regulated genes in Treg cells compared to conventional T cells. Whereas it is not clearly clarified in the paper from Mijnheer,G et al whether they use the up- and down- regulated genes in core Treg or they just used all differentially expressed genes as a prior gene set.

However, I checked the data sets from MSigDb, their prior datasets are normally seperated into up- and down- regulated genes.

How could I properly make a prior data set for GSEA analysis? Using all differentially expressed genes or separate the up- and down- regulated genes?

Thank you very much!

Reference:

Mijnheer, G., Lutter, L., Mokry, M. et al. Conserved human effector Treg cell transcriptomic and epigenetic signature in arthritic joint inflammation. Nat Commun 12, 2710 (2021). https://doi.org/10.1038/s41467-021-22975-7