Background We have an RNA-seq project on small intestine. The experimental design were like this: Four physiological states, each with three individuals (biological replicates), each individual with two samples (two parts of small intestine), thus 4 physiological states × three individuals × two parts. The reason we included two parts of same individual’s small intestines is that we want to take consideration of tissue heterogeneity, by pre-analysis of these replicates the difference between two parts is actually not significant.
Question Here is my question, when I applied a generalized linear model for the differential expression analysis on samples from two physiological states (e.g. DESeq2), should I treat samples as independent one thus 6 vs 6, or merge two samples from same individuals thus 3 vs 3. For the later one, can I regard it as technical replicate and merge reads from the two part together, or merge counts calculated from the two parts (e.g. collapseReplicates function in DESeq2). Or, should I include "individuals" as a blocking factor in the differential expression analysis.
There is an intensive discussion about biological replicates and technical replicates, like these Technical Or Biological Replicate Which step is better for merging technical replications? https://www.biostars.org/p/304008/#
but I am still confused about my situation, should I treat the two tissue parts from same individuals as biological or technical replicates.