Capturing Endogenous Long Noncoding RNAs and Their Binding Proteins Using Chromatin Isolation by RNA Purification.

Long noncoding RNAs (lncRNAs) exert their functions through binding to other RNA, genomic DNA, or proteins. The identification of proteins that associate with the lncRNA of interest sheds light on the molecular basis of its biological functions. This can be achieved by tagging the lncRNA with chemically modified ribonucleotides, or by using in vitro transcribed lncRNA to retrieve proteins from cell lysates. Alternatively, endogenous lncRNAs can be pulled down from cells or tissues with biotinylated antisense DNA oligonucleotides, which may overcome the potential pitfalls of using tagged lncRNAs, such as artifacts caused by tagging or non-physiological interactions. Here we describe the detailed protocol for chromatin isolation by RNA purification (ChIRP) from mammalian cell lines and mouse tissues, which captures endogenous lncRNAs and enables subsequent identification of their physiologically relevant binding partners.