Transposase Acting on an RNA/DNA Hybrid in Strand-Specific Sequencing

Strand-specific RNA sequencing is crucial for elucidating complex transcriptional regulation, yet existing methods often compromise between sensitivity, accuracy, and workflow simplicity. Here, we present directional SHERRY (d-SHERRY), a streamlined strand-specific RNA-seq method that leverages Tn5 transposase activity on RNA/DNA hybrids to eliminate second-strand cDNA synthesis while preserving strand-of-origin information. Through systematically optimizing reverse transcription and tagmentation conditions, d-SHERRY achieves over 95% strand specificity and detects more than 10,000 genes from as little as 100 pg of input RNA, outperforming commercial kits in library complexity and coverage uniformity. Its high directional precision enables accurate resolution of complex genomic regions, including overlapping antisense transcripts such as SLC4A5/MTHFD2, with over 98% specificity. With a hands-on time of just 0.5–1 h, d-SHERRY offers a rapid, sensitive, and reliable solution for strand-specific transcriptome profiling across a broad range of sample types and input amounts.