I am analyzing my small RNA seq data which contains 6 samples in total and includes two controls and 3 different treatments. In addition, one treatment has two different time points, so my controls also have two different time points. My coldata looks sth like this:<caption>colData</caption>
|treatment 1||4 h|
|sample 6||treatment 3||
Now I am wondering how to analyze the data. Should I set a reference level for the first ctrl and run the full dataset and later repeat the analysis using the second ctrl as a reference level, or should I split the dataset to each individual one by one comparison, or should I omit setting a reference level and simply use the contrast argument in the results function? Or is this a case where the multi-factor design function should be applied?
Of course, I know that this kind of analysis is only for exploratory purposes, as I do not have biological replicates included.
In the end, I would like to come up with a heat map showing the treatment differences compared to their respective controls. As I do not want to show all genes, I will have to order in a way to show the same genes for each condition, but on the other hand, I want to show the relevant genes for each condition. Any suggestions how to order/ cluster?
One last question: On a windows machine I didn't have problems with plotting the resLFC in an MA plot using "plotMA", whereas my mac seemed to have problems. The Error message was: "Error from the generic function 'plotMA' defined in package 'BiocGenerics': no S4 method definition for argument 'resLFC' of class 'numeric' was found"
Thank you in advance!