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Significance RNA sequencing is widely used to measure gene expression in biomedical research; therefore, improvements in the simplicity and accuracy of the technology are desirable. All existing RNA sequencing methods rely on the conversion of RNA into double-stranded DNA through reverse transcription followed by second-strand synthesis. The latter step requires additional enzymes and purification, and introduces sequence-dependent bias. Here, we show that Tn5 transposase, which randomly binds and cuts double-stranded DNA, can directly fragment and prime the RNA/DNA heteroduplexes generated by reverse transcription. The primed fragments are then subject to PCR amplification. This provides an approach for simple and accurate RNA characterization and quantification.

RNA sequencing by direct tagmentation of RNA/DNA hybrids