Protocol for analyzing tRNA-derived ncRNAs from small RNA-seq data using tRFUniverse functional analyses

Small RNA sequencing (smRNA-seq) reveals the complexity of tRNA-derived fragments (tRFs) in human physiology and disease. Here, we present a protocol for assessing tRF expression from smRNA-seq data. We describe steps for preprocessing, quality filtering, adapter trimming, and mapping reads. We then detail procedures for quantifying expressions and harmonizing data. We outline functional analyses using tRFUniverse, with a lung cancer case study from The Cancer Genome Atlas (TCGA) highlighting dysregulated tRFs and their roles. For complete details on the use and execution of this protocol, please refer to La Ferlita et al.¹.