TRACE‐seq: Rapid, Low‐Input, One‐Tube RNA‐seq Library Construction Based on Tagmentation of RNA/DNA Hybrids

Tn5 transposase has been widely used to simultaneously fragment and tag double-stranded DNA (dsDNA) with sequencing adaptors in library construction for next-generation sequencing. Recently, we demonstrated that Tn5 transposase also possesses tagmentation activity toward RNA/DNA hybrids, in addition to its canonical dsDNA substrates. Based on this new activity, we are able to skip multiple laborious and time-consuming steps in traditional RNA-seq methods, and rapid, low-input, cost-effective, one-tube RNA-seq library construction is thus enabled. The libraries constructed by Transposase-assisted RNA/DNA hybrids Co-tagmEntation (termed "TRACE-seq") demonstrate excellent performance in terms of gene expression measurement and differential gene expression analysis. Here, we present detailed protocols for TRACE-seq that will be broadly useful for the study of RNA biology and biomedical research. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Total RNA preparation Basic Protocol 2: TRACE-seq library construction Support Protocol: Tn5 transposome assembly.