Analysis of Long Noncoding RNA Chromatin Interactions by Chromatin Isolation by RNA Purification (ChIRP)

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Gene regulation plays a critical role in complex cellular processes such as development, differentiation, and cellular response to environmental changes. While the regulation of gene expression by transcription factors and epigenetic influences has been well studied over time, pervasive genomic transcription and the role of non‐coding RNAs in this process is a rapidly evolving field that remains to be thoroughly explored.Chromatin Isolation by RNA Purification (ChIRP) is a method that allows discovery of the sites of interaction of chromatin‐associated RNAs (e.g. lncRNAs) with genomic DNA sequences by using probe‐based hybridization to target RNA molecules in chromatin. DNA can be isolated from recovered chromatin and analyzed by quantitative PCR or next generation sequencing (ChIRP‐seq). Alternatively, RNA may also be isolated to detect other RNA molecules that may be associated with the RNA of interest.To enable the exploration of these RNA interactions in chromatin regulation, we have optimized the methods and developed ChIRP reagents. Using these reagents ChIRP experiments can be performed with reliable recovery of chromatin using lncRNA or other chromatin associated RNA as targets. Additionally, negative and positive control probe sets, and detection primers were developed to ensure first time success. We have performed ChIRP experiments with a HeLa cell lysate and capture oligos for the NEAT1 lncRNA. Isolated DNA was subjected to NGS library construction and sequenced on an Illumina HiSeq™ instrument. Sequences were aligned to the reference genome (hg19) and peaks were called. We successfully identified several NEAT1 binding sites in the genomic DNA sequence.In summary the methods developed allow unbiased discovery of RNA‐associated genomic DNA sequences, RNA sequences, and potentially proteins.