RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but solely relies on second strand cDNA synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analysis, to construct RNA-seq libraries without second strand synthesis. We discover that Tn5 transposome can randomly target the RNA/DNA heteroduplex and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to even single cells. SHERRY greatly reduces experimental difficulty, with higher reproducibility and GC uniformity than prevailing RNA-seq methods.