Recent genome-wide studies have yielded new insights into the biological function of long noncoding RNAs (lncRNAs), predominantly through analysis of their genomic addresses. These studies have revealed that a large number of lncRNAs map to regulatory elements in eukaryotic genome regions known as promoter and enhancer elements. Here, researchers from the University of Miami Miller School of Medicine review the principles of current methodologies for analyzing lncRNAs with high-throughput sequencing approaches.
- direct RNA sequencing,
- sequencing coupled with transcription, and
- isolation of protein complexes associated with lncRNAs followed by high-throughput sequencing.
Within these categories, they also describe detailed protocols for chromatin-associated RNA sequencing, nascent transcript Global run-on sequencing, and photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation.
Principle of RNA sequencing library preparation
Following removal of genomic DNA and ribosomal RNA, the remaining RNA is fragmented, and primers are added for cDNA synthesis. At this stage, polyadenylated transcripts can be enriched using oligo d(T)25 magnetic beads and oligo d(T)6 primers in the cDNA synthesis. To select for all RNA species, random hexamer primers are used. The PCR product is then ligated with sequencing adaptors, size-selected, and purified for sequencing.