I am using limma-voom for an RNA-Seq dataset with global down-regulation of gene-expression (experimentally confirmed). On top of that there is also a small set of genes in this dataset that is massively(!) up-regulated. I can't use RLE or TMM normalization because they normalize out the down-regulation.
I created a DGEList object and then set normalization factors to 1 (using edgeR's calcNormFactors function with method "none").
My question now is if there is still normalization by total library size happening if I proceed by applying the voom function and proceed as usual ... (or also alternatively analyze the data using edger). Or would I have to provide custom scaling factors to correct for library size?
Originally I had tried to set the scaling factors in the DGEList object to the ratio: (library size) / (median library size over all samples) but that didn't work as expected. So I got my doubts. As far as I got it the scaling factors are used in the model. Is the library size used separately (how do they play together then)?
I'd also be thankful for any general input about the best normalization option for a situation as described in the beginning.