title: Capture and sequencing of NAD-capped RNA sequences with NAD captureSeq.
abstract: Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture on streptavidin beads. Then, a highly efficient library preparation protocol tailored to immobilized, 5'-modified RNA is applied, with adaptor ligation to the RNA's 3' terminus and reverse transcription (RT) performed on-bead. Then, cDNA is released into solution, tailed, ligated to a second adaptor and PCR-amplified. After next-generation sequencing (NGS) of the DNA library, enriched sequences are identified by comparison with a control sample in which the first step of chemo-enzymatic biotinylation is omitted. Because the downstream protocol does not necessarily rely on NAD-modified but on 'clickable' or biotin-modified RNA, it can be applied to other RNA modifications or RNA-biomolecule interactions. The central part of this protocol can be completed in ∼7 d, excluding preparatory steps, sequencing and bioinformatic analysis.
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
authors.forename: Marie-Luise
authors.identifier: http://orcid.org/0000-0003-0740-8618
authors.initials: ML
authors.lastname: Winz
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
authors.forename: Hana
authors.initials: H
authors.lastname: Cahová
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Nübel
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Frindert
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Höfer
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
authors.forename: Andres
authors.initials: A
authors.lastname: Jäschke
mesh_terms: D019921:Biotinylation; D017931:DNA Primers / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D009243:NAD / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012315:RNA Caps / Q000235:genetics* / Q000378:metabolism*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D012315:RNA Caps; D009243:NAD