DESeq2 multiple treatments, multiple time points, multiple cell lines Note: this question has also been asked on Bioconductor Support
I know there are a lot of questions asking similar things here on this forum and I checked the vingette and did a lot of other research but I still have a hard time wrapping my head around it.
In my position, data is unfortunately just put in front of me and I'm asked for pretty pictures. Even making it clear to the wetlab people in the lab that we need replicates is an issue.
I have multiple cell lines, multiple time points and multiple treatments:
Cell lines: CL1, CL2, CL3 Time points: 6h, 24h Treatments: T1, T2, T3, Control
For each cell line and each time point, there are 3 different treatments plus a control. 3 replicates for each sample -&gt; 72 samples
What I want is actually quite simple; I want to measure control vs each treatment at each time point in each cell line. No testing across time points or across cell lines.
My first thought was to separate the data into 6 different data sets (CL1, 6h | CL1, 24h | CL2, 6h | CL2 24h | CL3, 6h | CL3, 24h) and simply do the DE analysis separately, but I read that this is not the way to go. In the end it would also be nice to get a normalized count matrix with all data normalized together, for PCAs and similar.
I hope I made it understandable. How do I design the DESeqDataSet?
To compare a subset of samples to another subset of samples, make one column of column data which will distinguish them all the way you want (so probably join cell line, treatment, timepoint), and make that column your design, and then when you call results, you specify the pair you want to contrast, one pair at a time.