Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq

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Ribonucleotides are the most common noncanonical nucleotides incorporated into the genome of replicating cells. They are efficiently removed by ribonucleotide excision repair initiated by RNase H2 cleavage. In the absence of RNase H2, such embedded ribonucleotides can be used to track DNA polymerase activity in vivo. To determine their precise location in Saccharomyces cerevisiae, we developed embedded ribonucleotide sequencing (emRiboSeq), which uses recombinant RNase H2 to selectively create ligatable 3'-hydroxyl groups, in contrast to alternative methods that use alkaline hydrolysis. EmRiboSeq allows reproducible, strand-specific and potentially quantitative detection of embedded ribonucleotides at single-nucleotide resolution. For the genome-wide mapping of other noncanonical bases, RNase H2 can be replaced with specific nicking endonucleases in this protocol; we term this method endonuclease sequencing (EndoSeq). With the protocol taking <5 d to complete, these methods allow the in vivo study of DNA replication and repair, including the identification of replication origins and termination regions.

Original languageEnglish
Pages (from-to)1433-1444
Number of pages12
JournalNature Protocols
Issue number9
Publication statusPublished - 27 Aug 2015


  • Embedded ribonucleotide
  • ribonucleotide misincorporation
  • genome-wide mapping
  • RNase H2
  • Ribonuclease H2
  • non-canonical nucleotide
  • base modification
  • endonuclease
  • library preparation
  • next generation sequencing
  • High-throughput sequencing
  • DNA replication
  • DNA polymerase


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