Mapping R-Loops and RNA:DNA Hybrids with S9.6-Based Immunoprecipitation Methods

Abstract

R-loops constitute a prevalent class of transcription-driven non-B DNA structures that occur in all genomes depending on both DNA sequence and topological favorability. In recent years, R-loops have been implicated in a variety of adaptive and maladaptive roles and have been linked to genomic instability in the context of human disorders. As a consequence, the accurate mapping of these structures in genomes is of high interest to many investigators. DRIP-seq (DNA:RNA Immunoprecipitation followed by high throughput sequencing) is described here. It is a robust and reproducible technique that permits accurate and semi-quantitative mapping of R-loops. A recent iteration of the method is also described in which fragmentation is accomplished using sonication (sDRIP-seq), which allows strand-specific and high-resolution mapping of R-loops. sDRIP-seq thus addresses some of the common limitations of the DRIP-seq method in terms of resolution and strandedness, making it a method of choice for R-loop mapping.

Figure 1:. Overview of the DRIP-seq and sDRIP-seq procedures.

Both approaches start by the same DNA extraction steps developed to preserve R-loops (RNA strands within R-loops are represented by squiggly lines). For DRIP-seq, the genome is fragmented using restriction enzymes, often resulting in kilobase-size fragments within which shorter R-loops are embedded. For sDRIP-seq, the genome is fragmented via sonication which results in smaller fragments and the shearing and loss of the displaced single-strand of R-loops (indicated by dashed lines). Following immunoprecipitation with the S9.6 antibody, DRIP leads to the recovery of three-stranded R-loops embedded within restriction fragments, while sDRIP recovers two-stranded RNA:DNA hybrids with little flanking DNA, ensuring higher resolution. For sDRIP, a library construction step must be included to convert RNA:DNA hybrids back to duplex DNA. As shown here, this is an opportunity to build strand-specific libraries. As detailed in the protocol itself, exogenous treatment with RNase H represents a key control for the specificity of both procedures; they are not shown here.

Figure 2:. Result of R-loop mapping strategies.

A. qPCR results from successful immunoprecipitations using the DRIP and sDRIP method (corresponding to qPCR check step 4.13). Results are from two independent experiments from human Ntera2 cells at a negative locus and two positive loci, including the highly R-loop-prone RPL13A locus and the moderately R-loop-prone locus TFPT. The y-axis indicates the yield of the immunoprecipitation as a percentage of the input DNA. Note that the recovery is slightly more robust for DRIP than sDRIP. B. The results of R-loop mapping conducted in human Ntera-2 cells are shown over a region centered around the CCND1 and neighboring ORAOV1 genes. The first two tracks correspond to DRIP-seq results, without and with RNase H treatment, respectively. The position of the restriction enzymes used to fragment the genome are shown at the top. The next six tracks represent the results of strand-specific sDRIP-seq, broken down between (+) and (−) strands (two replicates each) and pre-treated with RNase H, or not, as indicated. The last four track represents the results of R-loop mapping via the high-resolution strand-specific DRIPc-seq method (Sanz et al., 2016; Sanz and Chedin, 2019), where libraries are built from the RNA strands of R-loops. As can be clearly seen, the CCND1 and ORAOV1 genes lead to R-loop formation on the (+) and (−) strands, respectively, consistent with their directionality. RNase H treatment abolishes signal, as expected. C. Input DNA materials after restriction enzyme fragmentation (left) and sonication (right) are shown after the materials were separated by agarose gel electrophoresis. The DNA ladder corresponds to a 100 bp ladder and the 500 bp band is highlighted by an asterisk. D. XY signal correlation plots are shown to illustrate the reproducibility of sDRIP-seq (left), the overall sensitivity of sDRIP-seq to RNase H1 pre-treatment (middle), and the global correlation between sDRIP-seq and DRIPc-seq (right). All data from Ntera-2 human cells.