A colleague sent me a PCA plot last week. Three replicates per condition, control vs KO of a gene. PC1 cleanly separated replicate 1 from replicate 2 from replicate 3. PC2 separated control vs KO. The question that followed: should we run limma::removeBatchEffect() before differential expression?
The answer is “it depends,” and most of the time the answer is “no, do not pre-correct the matrix before DE testing.” But the question hides a few subtleties worth unpacking, because I have seen people both under-correct (ignore a real block effect) and over-correct (treat true biological variation as batch and squash it).