Find out shared differentially expressed genes between two cell lines due to knockdown treatment

Question

[Xinqi]

I have the following RNA seq data:

Hela cell, Trub1 KD
Hela cell, PUS7 KD
Hela cell, Scramble control
SHSY5Y cell, Trub1 KD
SHSY5Y cell, Scramble control

I want to know the RNA expression changes that are the same in Hela and SHSY5Y cells caused by Trub1 KD. Then I conducted the following statistical tests:

1. Hela Trub1 KD versus Hela Scramble control --> to observe the impact of Trub1 KD on Hela cells alone
2. SHSY5Y Trub1 KD versus SHSY5Y Scramble control --> to observe the impact of Trub1 KD on SHSY5Y cells alone
3. Hela (Hela Trub1 KD + Hela Scramble) versus SHSY5Y (SHSY5Y Trub1 KD + SHSY5Y Scramble) --> to observe the differences in RNA expression solely due to the difference in cell types.

After conducting these three sets of statistical tests, I plan to remove the genes that showed significant differences in group 3 from those in group 1. Similarly, I'll remove the significantly different genes in group 3 from those in group 2. This way, I can isolate the RNA expression differences that are purely due to the cell type.

The issue is, now I am a bit unsure about this third group. Should I include Trub1 KD or not?

Should it be Hela (Hela Trub1 KD + Hela Scramble) versus SHSY5Y (SHSY5Y Trub1 KD + SHSY5Y Scramble), or Hela (Hela Scramble) versus SHSY5Y (SHSY5Y Scramble), or Hela (Hela Trub1 KD + Hela PUS7 KD + Hela Scramble) versus SHSY5Y (SHSY5Y Trub1 KD + SHSY5Y Scramble)?

I hope my plan make sense. Any advice and help is greatly appreciated. Thanks!

Answer 1

[swbarnes2]

I'm not sure why you need the third comparison. If gene A changes due to the knockout in cell type A, but cell type B doesn't express that gene, you really want to throw that gene away? I don't see why that would help.

I'd go against the general rule in this specific case, and make each cell type its own DESeq object; I wouldn't try to compare them to each other. They are just way too different.

I'd take the gene lists from each KO vs scramble comparison, and sort out which genes are changed in both, and which are only changed in only one cell type.