Widespread DNA off-targeting confounds RNA chromatin occupancy studies

Several high-throughput sequencing methods have been used to study the genome-wide chromatin occupancy of long noncoding RNAs (lncRNAs), including ChIRP-seq, CHART-seq and RAP-seq. Many of the datasets obtained with these methods contain thousands of binding sites, which appears to be in contradiction with the low abundance of the interrogated lncRNAs. Here, we study the chromatin interactome of NESPR lncRNA in cells with varying levels of endogenous expression and perform a meta-analysis using dozens of RNA-chromatin interaction datasets in human and mouse cells. We demonstrate that thousands of regions reported to bind lncRNAs most likely arise from the spurious recovery of DNA elements, where the ends of the recovered DNA fragments exhibit partial complementarity with the probes used for the pulldown. In addition, crucial controls were rarely used in previous studies. Therefore, most chromatin regions reported as bound by trans-acting RNAs in recent studies in mammalian cells appear to be technical artifacts. We provide suggestions for assessing the quality of RNA-chromatin datasets and their improvement.