Hi everyone: I'm new to rna_seq data analysis. I'm going to analyze mRNA sequencing data from a certain cancer cell and drug treatment for DEGs using DESeq2.

The samples are prepared and sequenced as follows(every sample have two repeats):

1. cancer cells with nothing added;
2. cancer cells with tgfb only;
3. cancer cell with tgfb and compound a;
4. cancer cell with tgfb and compound b;
5. cancer cell with tgfb and compound c, (compounds a,b,c are inhibitors of tgfb);

My questions are:

1. How do I set up group information for DEGs ?
2. I planned the following combination:
   • tgfb+empty cell;
   • tgfb+compounds a,b,c;
   • empty+compounds a,b,c;

after this grouping, three analyses were performed to obtain the differential genes between the three combinations. Are these groups correct?

1. If empty cell is used as a control and grouped as four groups for one single analysis like: empty cell , tgfb, tgfb+a, tgfb+b, tgfb+c, does this make sense and how can the DEGs obtained be explained?

I am very confused, please help me.