Cycloheximide inhibits elongation by blocking the E-site which means we get an increased proportion of occupied A-sites. P-site tRNA cant move to E and A cant move to P so we get increased 28-29nt read populations. Flash freezing to inhibit translation would give a better picture of these empty A-site ribosome populations. This isn't my exact comfort zone so maybe refer to the following papers... https://pubmed.ncbi.nlm.nih.gov/24842990/ https://pmc.ncbi.nlm.nih.gov/articles/PMC6411040/