Optimizing murine sample sizes for RNA-seq studies revealed from large-scale comparative analysis

In order to determine the N, the usual approach is to perform a power calculation, which involves understanding the variability between samples and the expected effect size. Here, we focused on bulk RNA-seq experiments, which have become ubiquitous in biology, but which have many unknown or difficult to estimate parameters, and so the required analyses to determine the minimum N is typically lacking. We therefore performed two N=30 profiling studies between wild-type mice and mice in which one copy of a gene had been deleted, to determine how many mice would be required to minimize false positives and to maximize true discoveries found in the N of 30 experiment Dchs1+/- and Fat+/- heterozygous mice were generated using Regeneron’s (Tarrytown, NY) VelociGene® technology (ID #20561), and Wild-type littermates were used as controls. All experiments were performed on 100% C57BL/6NTac background. RNA-Seq was performed on RNA purified from heterozygous and homozygous Dchs1 or Fat4 heterozygous mice tissues (heart, kidney, liver, and lung).