Overview of RAP

RNA Antisense Purification (RAP) is a method to purify large non-coding RNA (lncRNA) complexes in vivo. RAP uses biotinylated antisense probes to hybridize to a target RNA to purify the endogenous RNA and its associated proteins, RNA, and genomic DNA from crosslinked cell lysate. We designed RAP to enable specific purification of chromatin associated with a target lncRNA, achieve high resolution mapping of the associated DNA target sites upon sequencing of the captured DNA, and robustly capture any lncRNA with minimal optimization. To achieve high specificity, RAP utilizes 120-nucleotide antisense RNA probes in order to form extremely strong hybrids with the target RNA thereby enabling purification using denaturing conditions that disrupt nonspecific RNA-protein interactions and nonspecific hybridization with RNAs or genomic DNA. To achieve high resolution, RAP uses DNase I to digest genomic DNA to ~150bp fragments, which provides high resolution mapping of binding sites. To robustly capture a lncRNA, RAP uses a pool of overlapping probes tiled across the entire length of the target RNA to ensure capture even in the case of extensive protein-RNA interactions, RNA secondary structure, or partial RNA degradation.