I work with RNA obtained by translating ribosome affinity purification. RNA is immunopurified from genetically labelled ribosomes, expressed only in the cell type of interest. An 'input' sample is taken, by extracting RNA directly from the homogenised tissue before immunopurification, as a background RNA comparison.
The first stage of analysis is to identify genes being translated in the cell type of interest. For this, most papers appear to use DESeq2 to compare their purified RNA with their input sample.
I have been concerned that the composition of the RNA between purified and input differs so much that DESeq2's normalisation factor calculation may confound the analysis. Depending on the cell type investigated, there may be between 4-9000 DEGs (out of 14000 with >10 counts average) between purified and input samples.
Currently the normalisation factors for purified samples are ~0.9 whereas for the input samples they are ~1.3. There were 4000 DEGs between purified and input.
Should I be concerned or is this not an issue? The libraries were prepared with ERCC spike-ins, in case this is recommended as an alternative option.