qDRIP: Quantitative differential RNA:DNA hybrid immunoprecipitation sequencing

Abstract

R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes and cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA:DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here we describe quantitative differential RNA:DNA immunoprecipitation (qDRIP), a method combining synthetic RNA:DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. Finally, we use these quantitative comparisons to make the first estimates of the absolute count of RNA:DNA hybrids per cell and their half-lives genome-wide. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights. real changes between conditions and even lead to misinterpretations (19)(20)(21). Outside of RNA:DNA hybrid sequencing, well-defined internal standards have proven to be a reliable tool to facilitate the accurate comparison of sequencing signal between experimental conditions (20-23). These spiked-in standards or "spike-ins" are introduced during sample preparation, carried through the experimental workflow, and ultimately sequenced to provide an internal normalization factor independent of total mapped reads. In ChIP-seq, Drosophila chromatin spike-ins have been used to quantitatively normalize sequencing signal from histone marks on the human genome, revealing a genome-wide depletion of these marks that was not apparent with normalization using total read counts (23). In RNA-seq, synthetic RNA spike-ins reduced bias in quantifying short genes (22) and changed data interpretations compared to standard normalization, thereby reconciling apparently contradictory experimental data (20).Because RNA:DNA hybrids can be created in vitro, pure, synthetic RNA:DNA hybrids are an ideal internal standard to normalize hybrid signal. Here, we describe quantitative differential RNA:DNA immunoprecipitation sequencing (qDRIP-seq), a method combining RNA:DNA hybrid internal standards with a high-resolution version of the strand-specific ssDRIP-seq protocol (13).We show that qDRIP-seq allows for sensitive, high-resolution, strand-specific mapping of RNA:DNA hybrids, and facilitates comparisons in hybrid content between different biological conditions. Results Design and preparation of RNA:DNA hybrid spike-insWe sought sequences for the RNA:DNA spike-ins that would serve as effective and orthogonal internal standards for qPCR and next-generation sequencing experiments in human cells. We chose E.coli sequences for the spike-ins, which had little homology to the human ge...