We have tried to sequence RNA from spent cell culture media using Smart-seq3. We have two groups across two time points, around 18 negative control samples which are just fresh media with no cells grown on them, and 4 water samples (RNA-free) which were subject to library preparation. Our idea was, we would use the negative control samples to identify and subtract background counts, and use Water samples to subtract any RNA signals generated by the reagents/contamination (As the water samples were processed in the same batch as rest of the samples).
However, from what I'm seeing this design seems rather unorthodox (but still logical to me).
We planned to perform the analysis with DESeq2 and other tools, however, from what I've read, the tools do not provide a provision to specify negative samples. What would be the most sensible way to approach this?