Small RNA-seq of an RNA mixture series for benchmarking of miRNA-seq methods
This study generated a ground-truth small RNA-seq dataset to benchmark normalization and differential expression methods for miRNA-seq analysis. Total RNA from mouse liver and spleen tissues obtained from five biological replicates was mixed in six defined proportions (0%, 20%, 40%, 60%, 80%, and 100% spleen), producing 30 biological samples that preserve known expected expression relationships while capturing realistic inter-individual variability. Additional technical replicates were included for each mixture level to assess technical noise and reproducibility. Libraries were prepared using standard small RNA sequencing protocols and sequenced to profile endogenous miRNA expression. Because the mixture ratios are known, the dataset enables objective evaluation of expression trends, fold-change accuracy, and differential expression performance across 15 pairwise comparisons. This resource was designed to provide a realistic alternative to simulations or spike-in controls and is publicly released to support future development, benchmarking, and comparison of miRNA-seq statistical methods.