Induro-seq to analyze subcellular enrichment of small RNAs.

Small non-coding RNAs, including microRNAs (miRNAs) and tRNA fragments (tRFs), play critical roles in gene regulation across diverse biological contexts. Although miRNAs and tRFs are traditionally viewed as cytoplasmic effectors, recent studies suggest they may also adopt functions within the nucleus. However, accurately mapping the subcellular localization of small RNAs remains technically challenging due to inherent biases in RNA yield between the nuclear and cytoplasmic compartments, as well as the presence of base-pair-disrupting RNA modifications. Here, we present a method for small RNA sequencing in example glioblastoma (GBM) cell lines that enables accurate subcellular localization by integrating defined synthetic spike-in controls and Induro-RT, a highly processive reverse transcriptase capable of reading through modified nucleotides. Spike-in controls correct for input disparities across compartments, while Induro-RT allows for the transcription of modified small RNAs, which are often overlooked by conventional reverse transcriptases. This approach enables unbiased detection of both canonical and modified small RNAs, providing a more accurate and comprehensive view of miRNA and tRF distribution between the nucleus and cytoplasm.