FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing.
Classical approaches to determine structures of noncoding RNA (ncRNA) probed only one RNA at a time with enzymes and chemicals, using gel electrophoresis to identify reactive positions. To accelerate RNA structure inference, we developed fragmentation sequencing (FragSeq), a high-throughput RNA structure probing method that uses high-throughput RNA sequencing of fragments generated by digestion with nuclease P1, which specifically cleaves single-stranded nucleic acids. In experiments probing the entire mouse nuclear transcriptome, we accurately and simultaneously mapped single-stranded RNA regions in multiple ncRNAs with known structure. We probed in two cell types to verify reproducibility. We also identified and experimentally validated structured regions in ncRNAs with, to our knowledge, no previously reported probing data.
Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, California, USA.
Jason G
JG
Underwood
Andrew V
AV
Uzilov
Sol
S
Katzman
Courtney S
CS
Onodera
Jacob E
JE
Mainzer
David H
DH
Mathews
Todd M
TM
Lowe
Sofie R
SR
Salama
David
D
Haussler
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods; D017931:DNA Primers; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006657:Histones / Q000235:genetics; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009474:Neurons / Q000502:physiology; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics*; D022661:RNA, Untranslated / Q000737:chemistry
D017931:DNA Primers; D006657:Histones; D022661:RNA, Untranslated; D012313:RNA