AbstractStructural analysis of RNA is an important and versatile tool to investigate the function of this type of molecules in the cell as well asin vitro. Several robust and reliable procedures are available, relying on chemical modification inducing RT stops or nucleotide misincorporations during reverse transcription. Others are based on cleavage reactions and RT stop signals. However, these methods address only one side of the RT stop or misincorporation position. Here, we describe Led-Seq, a new approach based on lead-induced cleavage of unpaired RNA positions, where both resulting cleavage products are investigated. The RNA fragments carrying 2′, 3′-cyclic phosphate or 5′-OH ends are selectively ligated to oligonucleotide adapters by specific RNA ligases. In a deep sequencing analysis, the cleavage sites are identified as ligation positions, avoiding possible false positive signals based on premature RT stops. With a benchmark set of transcripts inEscherichia coli, we show that Led-Seq is an improved and reliable approach based on metal ion-induced phosphodiester hydrolysis to investigate RNA structuresin vivo.