Hello,

I am computing a differential analysis on count data per region with DESeq2. For example, comparing only the region close to the transcription initiation site. Therefore, my count table represents the counts for each gene that map within a given interval (e.g. [TSS, TSS + 300]) for each sample.

I wonder if it is correct to compute the size factors with counts in the complete interval ([TSS, pA]) and then using them for the differential analysis limited in the region mentioned above. I would provide the size factors rather than calling estimateSizeFactors() with the data in my interval.

Thanks in advance for any insights

Essentially, it does not matter how you get the size factors as long as they're accurate and it's reproducible. A good diagnostic is MA-plots on the output of results(). They typically have an arrowhead-like shape and the tip if the arrowhead should be centered at y=0. Overall, the majority of points should be centered there as well. Checking control genes (if available) can help as well.