It is not yet possible to define the exact size and location of R-loop-forming regions on a genome-wide scale. In contrast, it is possible to determine the exact location of a specific R-loop-forming region in a specific gene using a previously developed bisulfite mapping approach (Yu et al., 2003). Essentially, this approach involved treating genomic DNA with bisulfite under non-denaturing conditions. Bisulfite specifically causes cytosine-to-uracil conversions in the single-stranded DNA portion of R-loops. On the other hand, cytosines in the RNA-DNA hybrid portion of the R-loop are protected from bisulfite conversion. Sequencing of both strands then revealed the location and strand in which cytosines were converted to uracils. The presence of an R-loop was identified by showing that the converted cytosines occurred primarily on one of the two strands of DNA (Yu et al., 2003). The location of bisulfite-induced conversions on a single strand of DNA was then used to define the boundaries of the R-loop at near-nucleotide resolution.

This use of bisulfite to detect single-stranded regions of DNA contrasts with the use of bisulfite in mapping 5-methylcytosine in DNA. For 5-methylcytosine mapping, DNA is denatured into single strands and then all cytosines are converted to uracils, while 5-methylcytosine is poorly converted (Frommer et al., 1992). Thus, while mapping 5-methylcytosine involves searching for unconverted cytosines, mapping single-stranded DNA involves detection of converted cytosines.

The use of bisulfite to map R-loops on a genome-wide scale poses several significant challenges. First, non-physiological R-loop formation, removal, expansion, or contraction can occur between the time of lysis and the time when bisulfite is used to mark single-stranded DNA (Kaback et al., 1979; Landgraf et al., 1996). Second, high sequencing depth is needed to search for converted cytosines across the entire genome (Sims et al., 2014). Finally, bisulfite causes infrequent, but measurable, background conversions in double-stranded DNA (Yu et al., 2003), which can generate a significant amount of noise on a genome-wide scale.

To map R-loops at near-nucleotide resolution, we developed a genome-wide bisulfite-based approach called bisDRIP-seq (Figure 1A). This method incorporates steps to overcome each of the challenges listed above. In this approach, cells are lysed in the presence of bisulfite and SDS. By including bisulfite during lysis, genomic DNA structures have as little time as possible to change conformation prior to the bisulfite modification of cytosines in single-stranded DNA regions. To overcome the problem of needing high sequence coverage, the S9.6 antibody (Boguslawski et al., 1986) is used to enrich for R-loops. The S9.6 antibody has high affinity for RNA-DNA hybrids and lower affinity for other double-stranded RNA sequences (Phillips et al., 2013). Thus, after genomic DNA is sheared using restriction digestion, the S9.6 antibody enriches the bisulfite-modified R-loops for sequencing analysis.

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Finally, to overcome the problem of stochastic cytosine conversions, a computational pipeline was developed to identify single-stranded regions using bisDRIP-seq data. This pipeline was developed to identify regions with high concentrations of cytosine-to-uracil conversions. This computational pipeline also identifies single-stranded regions that are likely to contain R-loops as opposed to other single-stranded DNA structures in the genome. Additionally, the pipeline was designed to reveal the specific strand orientation of R-loops.

Thus, bisDRIP-seq provides an approach to map R-loops at near-nucleotide resolution on a genome-wide scale.

We used bisDRIP-seq to map single-stranded DNA throughout the genome. Thirteen bisDRIP-seq experiments were performed on different samples of MCF-7 cells. After performing bisDRIP-seq on these samples, the DNA fragments were sequenced using a traditional post-bisulfite library preparation method (see Materials and methods).

We next aligned the sequenced reads to the genome using Bismark, an alignment approach typically used for 5-methylcytosine mapping (Krueger and Andrews, 2011). This was necessary since the conversion of cytosines to uracils would confound traditional read alignment programs. Bismark was used to map conversions associated with single-stranded DNA as follows: first, reads were aligned to the genome. Then the cytosines that had been converted to uracils were identified (Figure 1B). As expected, reads were detected that contained only a single conversion, consistent with noise due to low-level double-stranded DNA cytosine conversions. However, reads and regions were also observed that contained consecutive cytosine conversions (Figure 1—figure supplement 1A). These reads are suggestive of single-stranded DNA.

We next applied our bioinformatic pipeline to distinguish the multiple conversions seen in single-stranded DNA regions from the stochastic conversions due to background noise.

In the first part of this pipeline, the stochastic rate of conversions was estimated. This was estimated based on the overall cytosine-to-uracil conversion rate in a given sample. Reads with a high percentage of conversions were excluded from this calculation since it was assumed that those conversions were not stochastic.

Next, we generated a ‘bisDRIP-seq score’ for each read. This score was calculated based on the number of cytosines converted in the read, relative to the likelihood of observing that number of conversions by stochastic noise (see Materials and methods for more details). Next, the score was normalized so that the sum of bisDRIP-seq scores within each sample was the same.

Next, we calculated bisDRIP-seq scores for individual nucleotide positions in the genome. The bisDRIP-seq score for each nucleotide position was calculated as the sum of the bisDRIP-seq scores of all of the reads that overlapped with that nucleotide position. These scores provide a read-length resolution map of single-stranded DNA that filters out stochastic conversions and that is more comparable between genomic regions (Figure 1B).

Importantly, bisDRIP-seq scores were substantially reduced at specific gene loci in samples treated with RNase H, which degrades RNA in RNA-DNA hybrids (Figure 1—figure supplement 2). This supports the idea that elevated bisDRIP-seq scores reflect R-loops.

We next performed two basic tests of the quality of data produced by bisDRIP-seq:

First, we asked whether reads with high bisDRIP-seq scores are randomly distributed across the genome or whether they are clustered in specific regions. To simulate a random distribution, Monte Carlo simulations were applied to our data. Relative to these Monte Carlo simulations, bisDRIP-seq scores were found to be clustered in specific regions (Figure 1—figure supplement 1B–D).

Second, we performed correlation tests between the bisDRIP-seq scores obtained in each of our samples. In all cases, there was significant correlation between bisDRIP-seq samples (p<10⁻¹⁶, Spearman's rank-correlation test, Figure 1—figure supplement 3). 

We next wanted to know if bisDRIP-seq scores are associated with promoter regions that contain R-loops. R-loops were previously mapped to promoter regions (Ginno et al., 2012), where they are thought to play important roles in gene expression (Chen et al., 2015). These R-loops, as mapped using DRIP-seq, were found to be transcription dependent and correlated with gene expression (Sanz et al., 2016). We therefore wanted to determine if bisDRIP-seq scores have a similar enrichment and transcription dependence in active promoter regions.

In order to investigate promoter regions, we compiled a list of transcription start sites using the GENCODE database (Harrow et al., 2012) (see Materials and methods). We then defined promoter regions, for the purpose of our analyses, as the region one kilobase on either side of each of these transcription start sites.

We next performed several simple analyses to ensure that the results from bisDRIP-seq experiments recapitulate the promoter-region enrichment observed in DRIP-seq studies. First, bisDRIP-seq scores, like DRIP-seq reads, were found to be enriched in promoter regions relative to downstream exon-containing regions (Figure 2A and Figure 2—figure supplement 1A). Next, we found that the number of DRIP-seq reads correlates with bisDRIP-seq scores in individual promoter regions (p<2.2×10⁻¹⁶, Spearman's rank-correlation test, Figure 2—figure supplement 1B). Thus, bisDRIP-seq scores, like DRIP-seq reads, are enriched in promoter regions.

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bisDRIP-seq scores of promoter regions.

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We next asked if bisDRIP-seq enrichment in promoter regions depends on active transcription. To test if bisDRIP-seq enrichment requires transcription, bisDRIP-seq was repeated using MCF-7 cells treated with the transcription-inhibitor triptolide (Kupchan et al., 1972; Titov et al., 2011; Vispé et al., 2009). bisDRIP-seq enrichment was reduced in these samples (Figure 2—figure supplement 1C). This suggests that bisDRIP-seq enrichment in promoter regions depends on ongoing transcription.

We next asked if the bisDRIP-seq scores in promoter regions are correlated with promoter activity. Promoter activity was assessed using existing GRO-seq datasets from MCF-7 cells cultured in a similar manner to our cells (Hah et al., 2013; Hah et al., 2011). GRO-seq measures the presence of active polymerases on a genome-wide scale (Core et al., 2008). Thus, these GRO-seq datasets allow us to distinguish between promoter regions with low and high promoter activity. We therefore compared the number of GRO-seq reads with bisDRIP-seq scores in promoter regions (Figure 2—figure supplement 1D). In this analysis, GRO-seq-measured promoter activity correlates with bisDRIP-seq scores (p<2.2×10⁻¹⁶, Spearman's rank-correlation test). Thus, bisDRIP-seq scores are enriched in active promoter regions.

Notably, the enrichment of bisDRIP-seq scores in active promoter regions was reduced when RNA synthesis was blocked by triptolide treatment (Figure 2—figure supplement 1D).

Together, these results confirm that bisDRIP-seq scores, like DRIP-seq reads, are enriched in active promoter regions.

We next wanted to determine if the bisDRIP-seq score enrichment in promoter regions is due to co-transcriptional R-loops. Conceivably, cytosine conversions could occur if single-stranded DNA is exposed as a result of other single-stranded DNA structures near transcription start sites, including unwound DNA due to supercoiling (Hsieh and Wang, 1975), G-quadruplexes (Sen and Gilbert, 1988), or genomic regions that contain paused polymerases (Core et al., 2008) that become more accessible to bisulfite after SDS treatment.

Relative to other single-stranded DNA structures, R-loops are known to produce a specific cytosine conversion signature upon bisulfite treatment. Specifically, cytosine conversions are limited to one strand of DNA in an R-loop. In other types of genomic structures that expose single-stranded DNA, cytosines on both strands of DNA can be converted. Thus efforts to map R-loops with bisulfite must demonstrate preferential labeling of one strand of DNA (Yu et al., 2003).

Cytosine conversions can also occur as a result of two types of R-loops: ‘sense-strand R-loops’ and ‘antisense-strand R-loops.’ Sense-strand R-loops refer to R-loops in which the RNA component of the R-loop is transcribed from the annotated promoter in the expected direction. In these R-loops, the non-template strand of DNA is exposed for bisulfite-mediated conversion. Antisense-strand R-loops, on the other hand, contain RNA that is transcribed from the opposite DNA strand. In this case, the template strand of DNA for the canonical gene transcript would be modified by bisulfite. Thus, the specific strand of DNA that exhibits bisulfite-mediated cytosine conversions indicates if a sense-strand R-loop or antisense-strand R-loop is present.

In order to distinguish between R-loops and other single-stranded DNA structures, we took advantage of the cytosine conversion strand asymmetry that is expected to result from R-loops. In other types of genomic structures that expose single-stranded DNA, cytosines on both strands of DNA can be converted.

To determine if the observed bisDRIP-seq enrichment downstream of the transcription start site in promoter regions is caused by sense-strand R-loops, we repeated our comparison of bisDRIP-seq score with promoter activity. However, here bisDRIP-seq scores were plotted separately for the template and non-template strands of DNA (Figure 2B). With increasing promoter activity we observed increasing bisDRIP-seq scores on both strands, with preferential labeling of the non-template strand of DNA. Thus, although both strands of DNA had bisulfite-induced cytosine conversions, we also observed asymmetric labeling of the non-template strand (Figure 2C). This is consistent with a mixture of single-stranded DNA structure and sense-strand R-loops in active promoter regions (Figure 2D).

Notably, the higher bisDRIP-seq scores on the non-template strand than the template strand were largely eliminated in triptolide-treated samples (Figure 2B and C). This suggests that the sense-strand R-loops in the promoter region are transcription dependent.

The exact starting position and ending positions of promoter-associated R-loops remains unclear. This is due to the low resolution of conventional R-loop mapping methods (Chen et al., 2015; Ginno et al., 2012). We wanted to take advantage of the high resolution of bisDRIP-seq to map the exact boundaries of R-loops in promoter regions.

We first asked where R-loops are located in relation to transcription start sites. In many promoters, transcription initiates from multiple nearby transcription start sites (Carninci et al., 2006). This creates a practical limit to the precision that we can achieve in mapping the location of R-loops relative to transcription start sites.

We first mapped R-loops relative to all transcription start sites using metaplots of bisDRIP-seq scores. First, bisDRIP-seq scores were calculated for each nucleotide position surrounding the transcription start site in all individual promoter regions. Promoter regions were defined using the GENCODE database described above. Then, the bisDRIP-seq score at a given nucleotide position relative to the transcription start site was summed across all promoter regions. These scores were then plotted separately for the non-template strand and the template strand (Figure 3A).

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The resulting metaplot suggests that a mix of R-loops and other single-stranded structures surround the transcription start site. The presence of single-stranded DNA at transcription start sites is suggested by the peak of bisDRIP-seq scores near the transcription start site (Figure 3B). The location of these single-stranded structures is consistent with previous maps of single-stranded DNA (Kouzine et al., 2013). However, the presence of R-loops is specifically suggested by asymmetric, preferential labeling of the non-template strand (Figure 3B). Indeed, the non-template strand bisDRIP-seq scores are significantly higher than the template strand bisDRIP-seq scores immediately 3’ of the transcription start site (p<0.005, Wilcoxon signed-rank test) (Figure 3A, Figure 3—figure supplement 1B). This suggests that the transcription start site is the 5’ boundary of promoter-associated R-loops.

We next asked whether the R-loops bounded by the transcription start site are dependent on transcription. We repeated our metaplot analysis using the samples treated with triptolide. In these samples, there was minimal difference between the template and non-template strand bisDRIP-seq scores (Figure 3A). The small remaining difference in scores upon triptolide-treatment may reflect a real difference, but it may also reflect noise in our measurements. Overall, the loss of bisDRIP-seq score strand asymmetry upon triptolide treatment demonstrates that the enrichment of R-loops 3' of the transcription start site requires transcription.

The transcription start site boundary was even more apparent after applying a background correction. To do this, we defined an ‘R-loop signal,’ which reflects the difference in bisDRIP-seq labeling of the non-template strand from the template strand after using the triptolide-treated samples for background correction. Thus, the template strand metaplot from the triptolide-treated sample was subtracted from the template strand metaplot from the control sample. The same background correction was used for the non-template strand metaplot. This background correction further enhances the demarcation of the transcription start site as the 5’ boundary of promoter-associated R-loops (Figure 3C). Next, we subtracted the template strand metaplot from the non-template metaplot to generate a metaplot of the R-loop signal (Figure 3D). In this plot, the 5’ boundary of R-loop signal at the transcription start site is very pronounced.

We repeated this analysis on promoters that have either high promoter activity or low promoter activity (Figure 3E and F, Figure 3—figure supplement 1C, Figure 3—figure supplement 2). A peak of R-loop signal was only observed immediately 3' of the transcription start site in the analysis of active promoters. This is consistent with our previous analysis showing that R-loops specifically form 3' of the transcription start site in active promoters.

Notably, the observed difference in strand bisDRIP-seq scores 3' of the transcription start site in active promoters is lost after RNase H treatment (Figure 3—figure supplement 3). This confirms that the R-loop signal observed in active promoter regions is caused by R-loops.

Taken together, these data indicate that the transcription start site demarcates the 5’ boundary of promoter-associated R-loops. Additionally, since only noise was observed from promoter regions with low promoter activity, these regions were removed from future analysis unless otherwise noted.

We next wanted to know if there is a 3’ boundary to R-loops. Based on the metaplot analysis in Figure 3D,R-loop signal drops approximately 200–250 bp downstream of the transcription start site. This is further from the transcription start site than the typical first post-transcription start site nucleosome (Schones et al., 2008) and the location of promoter-proximal RNA polymerase II pausing (Core et al., 2008), suggesting that these features probably do not impede R-loop expansion. On the other hand, 200–250 bp is reasonably close to the median distance between the transcription start site and the first exon-intron junction, which is 181 bp in our dataset (see Materials and methods). Also, previous studies found that knockdown of the 5' splice site-binding factor SRSF1 induces the formation of R-loops (Li and Manley, 2005), which could be explained if splicing is involved in bounding R-loop expansion. These pieces of evidence suggested that the first exon-intron junction might act as the 3' boundary to R-loop expansion in promoter regions.

We therefore asked where R-loops are located relative to the first exon-intron junction. We used the 5' end of the first intron as the reference point for a metaplot of bisDRIP-seq scores. Intronless genes were not considered in this analysis. In these metaplots, we observed that the bisDRIP-seq scores on the non-template strand are significantly higher than on the template strand immediately 5' of the first exon-intron junction (Figure 4A and Figure 4—figure supplement 1A). This strand asymmetry in bisDRIP-seq scores drops 3' of the exon-intron junction. This suggests that the 3’ end of R-loops are bounded by the first exon-intron junction.

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Location of exon-intron junctions analyzed in Figure 4.

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We further tested the idea that the first exon-intron junction is the 3’ boundary of R-loops. To do this, we asked if the size of the R-loop-forming region in promoter regions increases with the length of the first exon. To test this question, metaplot analysis was repeated on groups of promoter regions with different sized first exons. First, promoter regions were binned into five groups based on the annotated size of the first exon. Then, metaplots were created of the R-loop signal centered around either the transcription start site or the first exon-intron junction (Figure 4B). Strikingly, in groups of promoter regions with longer first exons, the R-loop signals are also longer. This supports the idea that R-loops are bounded at both the transcription start site and the first exon-intron junction.

We next asked if we could map the location of the 3' R-loop boundary at near-nucleotide resolution. bisDRIP-seq scores are shared across entire reads, which limits the resolution of using bisDRIP-seq scores to read length resolution. On the other hand, cytosine-to-uracil conversions should map R-loops at near-nucleotide resolution. We therefore generated a metaplot of the strand asymmetry of cytosine conversions relative to the first exon-intron junction. First, the number of conversions on either strand were background corrected by subtracting the number of conversions observed in our triptolide bisDRIP-seq data. Next, the log ratio of conversions on the non-template strand relative to the template strand was plotted at each site relative to the exon-intron junction (Figure 4C). In this metaplot there is a decrease in the relative number of conversions on the non-template strand within base pairs of the exon-intron junction (Figure 4—figure supplement 1B). Thus, it appears that the R-loop boundary is within a few base pairs of the first exon-intron junction.

We next asked whether other exon-intron junctions also act as R-loop boundaries. In particular, we focused on the junctions between the first intron and the second exon, as well as the junction between the second exon and the second intron. We repeated our metaplot analysis by plotting R-loop signal at each position relative to the given exon-intron or intron-exon junction (Figure 4—figure supplement 1A). No clear peak in R-loop signal is observed near these downstream intron-exon junctions. This suggests that only the first exon-intron junction acts as a boundary to R-loop formation.

Together, these results suggest that there is a boundary to R-loop formation located within base pairs of the first exon-intron junction. 

We noticed that there is negative R-loop signal upstream of the transcription start site (Figure 3D and F). This could be caused by ‘antisense-strand R-loops,’ i.e., with antisense RNA transcripts hybridized to the annotated non-template strand of DNA (See Figure 3—figure supplement 4C for this structure). Antisense-strand R-loops would result in more prominent bisulfite conversions on the annotated template strand. This type of labeling is opposite from the non-template strand labeling that is caused by the predominant type of R-loop that forms from sense transcription.

We considered that antisense transcription could lead to antisense-strand R-loops that generate these negative R-loop signals. To test this, we calculated the antisense-transcription activity of each promoter region upstream of the transcription start site using the previously described GRO-seq dataset (Hah et al., 2013). In promoter regions with high antisense-transcription promoter activity, the template-strand bisDRIP-seq scores upstream of the transcription start site were significantly higher than on the non-template strand (Figure 3—figure supplement 4A and B). These data indicate a correlation between antisense transcription and antisense R-loops.

We next used the promoters that showed the highest level of antisense-transcription promoter activity to generate a metaplot of R-loop signal. In this metaplot, the negative R-loop signal was prominent upstream of the transcription start site (Figure 3—figure supplement 4D). Taken together, these data suggest that some promoter regions contain antisense-strand R-loops and that this is linked to antisense transcription in these promoter regions.

We next wanted to identify the promoters that show the strongest association with transcription-dependent R-loops. Any unique features associated with these promoters may be directly related to the R-loops forming at these promoter regions. We searched for promoter regions with two major features: First, we searched for promoter regions that showed disproportionately high bisDRIP-seq score on the non-template strand compared to the template strand. Second, we searched for promoter regions where the majority of the bisDRIP-seq score on the non-template strand was lost upon triptolide treatment. In this analysis, we noticed a set of promoter regions that exhibited both of these features (Figure 5A). We therefore ranked genes based on the sum of these two features to identify the genes that show the strongest association with R-loop structures (Table 1).

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R-loop scoring of promoters studied in Figure 5A, B.

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Two important classes of genes were identified in this analysis and both lack introns. First, six of the top 25 genes (24%) and nine of the top 50 genes (18%) are replication-dependent histone genes. The second class of genes encode intronless noncoding RNAs, including MALAT1, NEAT1, RPPH1, and CTB-58E17.1. We noticed that two other top hits, the protein-coding genes RHOB and JUNB, are also intronless genes.

In general, intronless genes appear to be enriched among the promoters that show strong association with R-loop structures. In total, 44% of the top 25 genes in our list are intronless, compared to approximately 2% of long non-coding RNAs (lncRNAs) and 3% of protein-coding genes (Derrien et al., 2012; Louhichi et al., 2011). The presence of R-loops in the promoter regions of these intronless genes suggests that the first exon-intron junction does not promote R-loop formation.

The presence of R-loops in the replication-dependent histone genes is potentially interesting given how these genes are regulated. As their name suggests, these histone genes are regulated in a cell-cycle dependent manner (Robbins and Borun, 1967). They also lack poly-A tails and are processed in special histone bodies in the nucleus (Dominski and Marzluff, 1999). Thus these genes are co-regulated using special processing pathways.

We first considered the possibility that the prominent R-loop signal detected in histone promoter regions simply reflects high promoter activity in these genes. However, histone genes consistently had higher R-loop-associated bisDRIP-seq scores than the majority of genes with similar promoter activity (Figure 5B). This suggests that promoter activity does not explain the strong R-loop signal observed in histone genes.

Similar analysis indicated that nuclear RNA levels and recruitment of RNA polymerase II also do not explain the R-loop signal observed in histone genes (Figure 5—figure supplement 1A and B).

We next examined the prevalence of R-loops and other single-stranded DNA structures in the promoter regions of histone genes. We repeated our metaplot analysis using the bisDRIP-seq scores from the entire class of replication-dependent histone genes (Figure 5C). In the resulting metaplot, bisDRIP-seq scores are low on the template strand and in triptolide-treated samples. This suggests that there is little single-stranded structure outside of R-loops in the promoter regions of these genes. In contrast, bisDRIP-seq scores are high downstream of the transcription start site on the non-template strand. Moreover, these high bisDRIP-seq scores are not observed after RNase H treatment (Figure 5—figure supplement 1C–E). Together, these results indicate that histone genes, as a group, have a high level of R-loop formation relative to the formation of other single-stranded DNA structures.

We next asked whether R-loops are bounded in replication-dependent histone genes. It was not clear if R-loops would be bounded in these genes since they lack introns and therefore they lack exon-intron junctions (Marzluff et al., 2008). Conceivably, the R-loops could extend to the entire length of the transcript. We therefore identified the boundaries of the R-loop signal in each of the nine histone genes that had the highest propensity to form R-loops (Figure 5A). As expected, the 5’ boundary of the R-loops appear to be near the transcription start site in all nine genes. In five of the nine histone genes, the entire R-loop appeared to be restricted to the initial portion of the gene (Figure 5D and Figure 5—figure supplement 2A–E). Sequence analysis of these boundaries does not reveal a clear sequence enrichment or motif (Figure 5—figure supplement 1D and E), making it currently unclear how this boundary is determined. In other cases, like HIST1H1E, R-loops seemed to cover nearly the entire gene (Figure 5E and Figure 5—figure supplement 2F–I). Together these results suggest that additional factors may establish 3’ R-loop boundaries in a subset of the replication-dependent histone genes.

Another set of genes which preferentially exhibit R-loops in their promoter regions are MALAT1 and NEAT1. MALAT1 and NEAT1 are adjacent genes that encode abundant, intronless lncRNAs (Hutchinson et al., 2007). These lncRNAs remain in the nucleus where they are involved in the regulation of transcription (Hirose et al., 2014) and splicing (Tripathi et al., 2010), respectively. Both MALAT1 and NEAT1 are longer than 3 kb, which is longer than the replication-dependent histone genes studied above. We were therefore interested in whether there are boundaries to R-loop expansion in these much longer intronless genes.

We first asked where R-loops are located in MALAT1 and NEAT1. The R-loop forming region in MALAT1 extends from the transcription start site to a position approximately 1700 bp downstream, with a sharp decrease in R-loop signal downstream of this position (Figure 6A). Similarly, the R-loop in NEAT1 extended approximately 1400 bp from the transcription start site (Figure 6B). Beyond this site, there was minimal detectable R-loop signal. As with the R-loops in the replication-dependent histone genes, these R-loops showed nearly complete loss of bisDRIP-seq signal on the non-template strand after triptolide treatment (Figure 6—figure supplement 1A and B). Moreover, the high bisDRIP-seq scores in this region are not observed after RNase H treatment (Figure 6—figure supplement 2A and B). This suggests that relatively long R-loops form in MALAT1 and NEAT1 and that these R-loops are bounded to the 5' end of each gene.

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Although our mapping reveals the location of the R-loops in MALAT1 and NEAT1 at near-nucleotide resolution, analysis of previous DRIP-seq datasets (Sanz et al., 2016) reveal signals in the same overall regions (Figure 6—figure supplement 1A and B).

Interestingly, there appears to also be periodicity in the bisDRIP-seq scores on the non-template strand of NEAT1 with peaks and valleys every 300 base pairs (Figure 6B). This phenomenon is also detectable, but less prominent in MALAT1 (Figure 6A).

These valleys may indicate the existence of smaller R-loops in some MALAT1 or NEAT1 genes in some cells. This idea is supported by examining individual reads within the R-loop forming region in MALAT1. In most cases, we observed that cytosines were almost completely converted in individual reads (Figure 6—figure supplement 3A). However, we also observed a subset of reads with long stretches of cytosine conversions on one end and long stretches of unconverted cytosines on the other end (Figure 6—figure supplement 3B). The region where the conversions stop occurring in individual reads might reflect an internal border of a R-loop. It should be noted that we cannot exclude the possibility that this may reflect the location of a structured region in the single-stranded DNA that prevents bisulfite reactivity. Nevertheless, the presence of peaks and valleys within the R-loop-forming region of NEAT1 and MALAT1 raises the possibility of heterogeneity in the size and location of the individual R-loops within the larger R-loop forming region identified by bisDRIP-seq.

Previous efforts to map R-loops have primarily relied on immunoprecipitation of RNA-DNA hybrids (Chédin, 2016). Traditionally, DNA fragments containing an R-loop are recovered and sequenced. The sequenced fragments contain both the DNA involved in the R-loop and regions of DNA that are not in the R-loop. Newer approaches, like DRIPc-seq (Chen et al., 2015; Sanz et al., 2016), can provide higher resolution by sequencing the RNA component of the R-loop. We therefore next wanted to determine if the specific R-loop boundaries detected by bisDRIP-seq could also be detected in the human DRIPc-seq datasets.

We first compared the location of R-loop signal in metaplots of DRIPc-seq and bisDRIP-seq signal in active promoters regions. As we demonstrated earlier, the R-loop signal in bisDRIP-seq is bounded between the transcription start site and the first exon-intron junction (Figure 7A). Thus, we observe a tight peak of R-loop signal within a few hundred base pairs of the transcription start site in active promoter regions. On the other hand, DRIPc-seq shows a marked enrichment of reads following the transcription start site; however, there is no clear boundary of sense-strand DRIPc-seq reads peaks within 5 kb of the transcription start site (Figure 7A). This highlights the improvement in resolution obtained by bisDRIP-seq.

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We next compared the R-loop maps generated using DRIPc-seq or bisDRIP-seq at individual gene loci. In some genes, such as MALAT1 and NEAT1, we observed tight concordance between the R-loop maps generated from DRIPc-seq and bisDRIP-seq (Figure 7B and C). However, the boundaries demarcated by bisDRIP-seq appear more clear than the boundaries demarcated by DRIPc-seq. On the other hand, there were few DRIPc-seq reads mapped to genes such as RPPH1 and HIST1H2BG, which displayed strong R-loop signal in bisDRIP-seq (Figure 7D and E). This suggests the possibility that bisDRIP-seq might identify a set of R-loops that are not observable by DRIP-seq.

We next asked if the size or location of the R-loop might change ex vivo. bisDRIP-seq labels R-loops upon cell lysis, while other mapping methods recover the R-loop hours or days after cell lysis. R-loops could conceivably, expand, contract, or disappear during this time period. This seemed plausible, since the structure of some R-loops has previously been observed to change in solution (Kaback et al., 1979; Landgraf et al., 1996). To test this possibility, we performed a ‘delayed-bisulfite’ bisDRIP-seq experiment. In this experiment, we delayed the bisulfite step of our protocol so that samples were only treated with bisulfite 16 hr after cell lysis. In some genes, such as MALAT1 and NEAT1, the bisDRIP-seq scores were similar when using the delayed-bisulfite protocol. In other cases, the delayed-bisulfite treatment was associated with a marked depletion of the R-loop signal in RPPH1 and HIST1H2BG (Figure 7B–E). This suggests the possibility that some R-loops may not be stable for prolonged periods of time ex vivo and therefore may not be observed in approaches that do not label or recover R-loops during lysis.

MCF-7 cells were used for all experiments. Cell lines were originally obtained from ATCC. Cell identification was performed by ATCC, which identifies cell lines using STR profiling, and cell identity was regularly checked by visual inspection of morphologies. Independently prepared vials of cell lines were tested for mycoplasma by Hoechst staining; these cell lines were tested after the conduction of the experiments described here.

MCF-7 cells (10 million per 15 cm tissue culture dish) were cultured for three days at 37°C in 50 ml of Gibco's phenol red-free Dulbecco's Modified Eagle's Medium (Thermo Fisher Scientific, Waltham, Massachusetts) containing 5% charcoal-stripped fetal bovine serum (Thermo Fisher Scientific, Waltham, Massachusetts). Media was replaced after two days. In these experiments, we included MCF-7 cells treated for different amounts of time with 100 µM estrogen (Sigma-Aldrich, Saint Louis, Missouri) prior to lysis to ensure transcriptional activation at a broad set of genes (Hah et al., 2011). The use of estrogen in some samples ensures that we can maximally capture R-loops that occur in either basal and stimulated conditions.

Each of the thirteen control-treated bisDRIP-seq experiments and each of the two triptolide-treated bisDRIP-seq experiments were performed using a separate dish of MCF-7 cells. Thus, each bisDRIP-seq replicate used a different sample of MCF-7 cells that had, at a minimum, been cultured on a separate dish for three days.

In the bisDRIP-seq protocol, R-loops are treated with bisulfite during cell lysis under non-denaturing conditions. The basic concept underlying this approach is that bisulfite can interact with single-stranded DNA, but cannot interact with DNA in a double helix (Yu et al., 2003). This contrasts with 5-methylcytosine mapping, which is performed under denaturing conditions that cause all double-stranded DNA to be single stranded and susceptible to bisulfite treatment. It is therefore worth noting that bisDRIP-seq could be affected by 5-methylcytosine. If 5-methylcytosine is present in the single-stranded DNA of the R-loop, there will be minimal conversion at those sites. This should not substantially impact R-loop mapping, since 5-methylcytosines are found only in a CpG sequence context and most R-loops will have some cytosines that are not followed by guanine. Nevertheless, this issue could be considered during data analysis.

Prior to performing bisDRIP-seq, lysis buffer was prepared containing 55.5 mg of ammonium sulfite monohydrate (Sigma-Aldrich, Saint Louis, Missouri), 760 µl of 45% ammonium bisulfite (Pfaltz and Bauer Inc, Waterbury, Connecticut), 85.5 µl of 20 mM hydroquinone (Sigma-Aldrich, Saint Louis, Missouri) and 62.7 µl of 0.5 M ethylenediaminetetraacetic acid (EDTA) in a final volume of 2.85 ml. Lysis buffer was prepared using degassed water.

Additionally, ammonium bisulfite solution was prepared containing 0.67 g ammonium sulfite monohydrate, 2.08 g of sodium bisulfite (Sigma-Aldrich, Saint Louis, Missouri), 5 ml of 45% ammonium bisulfite in a final volume of 6 ml. This ammonium bisulfite solution was prepared using degassed water, following the methodology described by Hayatsu et al., 2004.

Immediately prior to cell lysis, media was removed from cells. Cells were washed three times in ice-cold PBS. Cells were then scraped off of plates into ice-cold PBS. These cells were then transferred to a 15 ml conical tube. The conical tube containing cells was centrifuged for 3 min at 300 g . PBS was then removed from cells.

Next, cells were lysed in the presence of bisulfite. Bisulfite was included in the lysis buffer to achieve rapid single-stranded DNA modification that would minimize R-loop expansion or contraction. First, lysis buffer (1.5 ml) was added to the cells. Cells in lysis buffer (475 µl) were added to each of two microcentrifuge tubes, each of which contained 25 µl of lithium dodecyl sulfate. Samples were then incubated in a shaking incubator for 30 min at 37°C at 1100 rpm, while being protected from light.

Next, the concentration of bisulfite in samples was increased in order to increase the rate of the irreversible second hydrolytic deamination step in the bisulfite conversion process (Hayatsu et al., 2004). The concentration of bisulfite was increased by transferring samples to a microcentrifuge tube containing 1 ml of ammonium bisulfite solution and 26.8 µl of 20 mM hydroquinone. Samples were then incubated in a shaking incubator for 2 hr at 37°C at 1100 rpm, while being protected from light.

Next, bisulfite was removed from the samples through dialysis. First, samples were added to 2.5 ml of dialysis buffer (50 mM Tris pH 8, 10 mM EDTA) in a 15 ml conical centrifuge tube. Samples were then added to an Amicon Ultra-4 Centrifugal Filter Unit with a 30,000 nominal molecular weight limit (EMD Millipore, Darmstadt, Germany). Next, the samples were centrifuged following the instructions of the centrifugal filter unit manufacturer. After centrifugation, fresh dialysis buffer was added to the centrifugal filter unit and centrifugation was repeated. This dialysis process was repeated until the sample was nominally dialyzed at least 200 fold. Proteinase K buffer (50 mM NaCl, 50 mM Tris pH 8, 10 mM EDTA) was then added to the sample and centrifugation was repeated. This dialysis process was repeated until the sample was nominally dialyzed at least 5000 fold in Proteinase K buffer. At this point, the samples were resuspended in 1 ml of Proteinase K buffer and transferred to a microcentrifuge tube.

Next, we used Proteinase K (Life Technologies, Carlsbad, California) to degrade the proteins in our samples. In order to treat our samples with Proteinase K, 50 µl of 20% SDS and 200 µl of 20 µg/µl Proteinase K was added to each sample. Samples were then incubated overnight at 37°C while being rotated end over end.

Nucleic acids were purified from any remaining proteins present in the sample using phenol-chloroform. Samples were split into two microcentrifuge tubes. An equal volume of Phenol-chloroform (Life Technologies, Carlsbad, California) was added to each sample. Samples were mixed and then centrifuged for five minutes at 18400 g. The aqueous phase of each sample was transferred to a new microcentrifuge tube. Each sample was then combined with an equal volume of chloroform. Samples were mixed and centrifuged for two minutes at 18400 g. The aqueous phase of each sample was transferred to a new microcentrifuge tube. Samples were then ethanol precipitated for 2 hr at 4°C. The precipitated DNA was then transferred and combined in a new tube by swirling the DNA around a pipette tip. Finally, the DNA was washed with 70% ethanol.

Next, the DNA was fragmented using restriction enzymes. The precipitated DNA was resuspended in 850 µl of NEB buffer 3.1 (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 100 µg/ml bovine serum albumin). The samples were then digested overnight at 37°C by a cocktail of restriction enzymes, which included: 200 U/ml HindIII, 200 U/ml EcoRI, 100 U/ml BsrGI, 200 U/ml XbaI and 50 U/ml SspI (all from New England Biolabs, Ipswich, Massachusetts) in a total volume of 900 µl.

Next, the quality of the DNA digest was assessed and the concentration of DNA was measured in preparation for the RNA-DNA hybrid immunoprecipitation. DNA digest efficiency was measured by running the DNA on a 0.8% agarose gel stained with ethidium bromide. This was performed for every bisDRIP-seq experiment and samples were only used for immunoprecipitation if the digested DNA was mostly between 1 kb and 10 kb long. The concentration of DNA was measured using a Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts) and a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, California) following the instructions provided by the Quant-iT dsDNA Assay kit manufacturers.

Next, RNA-DNA hybrids were enriched using an immunoprecipitation protocol modeled after the DRIP-seq methodology described by Ginno et al. (2012). Digested DNA (20 µg) was added to 20 µg of S9.6 antibody (Kerafast, Boston, Massachusetts) in 1 ml of immunoprecipitation buffer (10 mM sodium phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100). The sample was then incubated overnight at 4°C while being mixed end over end. Next, the sample was added to 150 µl of Dynabeads Protein G (Life Technologies, Carlsbad, California) washed in immunoprecipitation buffer. Samples were incubated for 2 hr at 4°C while being mixed end over end.

Next, the Dynabeads Protein G and immunoprecipitated DNA were washed. First, the supernatant was removed after applying the sample to a magnet for 3 min. Next, beads were washed three times in 750 µl of immunoprecipitation buffer. Each wash lasted 10 min and during the wash the samples were mixed end over end at 4°C. On each occasion, the supernatant was removed after applying the sample to a magnet for 3 min.

Next, the bisulfite reaction was completed. First, we added 150 µl of 0.3 N NaOH to the samples. The samples were then incubated for 20 min at 37°C. This step should complete the bisulfite reaction. We then neutralized the NaOH by adding 150 µl of 0.3 N HCl and 17.5 µl of 1 M Tris pH 8.0.

Next, we eluted the immunoprecipitated DNA. First, 0.5 µl of 100 U/µl RNase I was added to each sample. Samples were then incubated for 20 min at 50°C. RNAs still bound to DNA were degraded by adding 1 µl of 0.5 M MgCl₂ and 3 µl of 5 U/µl RNase H (New England Biolabs, Ipswich, Massachusetts) to each sample. The samples were then incubated for 20 min at 37°C. To ensure that all DNA fragments were eluted, we added 2 µl of 20 µg/µl of Proteinase K to each sample. The samples were then incubated for 1 hr at 50°C. Finally, a magnet was added to each sample for 3 min and the supernatant containing the eluted DNA was transferred to a new microcentrifuge tube.

Next, DNA was extracted from the supernatant using phenol/chloroform. Prior to adding phenol/chloroform, 150 µl of water was added to each sample. Samples were then combined with 500 µl of phenol/chloroform. Samples were mixed thoroughly and then centrifuged at 18400 g for 5 min. 400 µl of the aqueous phase was transferred to a new microcentrifuge tube and then 400 µl of chloroform was added to this aqueous phase. These samples were mixed and then centrifuged at 18400 g for 2 min. The supernatant was transferred to a new microcentrifuge tube. Finally, the DNA in the sample was ethanol precipitated in the presence of 20 µg of glycogen for 48 hr.

In order to treat cells with triptolide (R and D Systems, Minneapolis, Minnesota), 20 mM triptolide was prepared in dimethyl sulfoxide.

Triptolide was added to the media covering the MCF-7 cells at a concentration of 1 µM. The plates containing the cells were gently swirled to evenly distribute the triptolide. The plates were then incubated for 2 hr at 37°C. Following this triptolide treatment, bisDRIP-seq was performed as described above.

Each of the two triptolide-treatment biological replicates was performed on a separate plate of cells.

Two input bisDRIP-seq samples were prepared using the bisDRIP-seq protocol until the S9.6 antibody immunoprecipitation step. Instead of adding the digested DNA to S9.6 antibody, 15 µl of 3 N NaOH was added to 0.2 µg of digested DNA in 135 µl of water. The samples were then incubated for 20 min at 37°C. This step should complete the bisulfite reaction. We then neutralized the NaOH by adding 150 µl of 0.3 N HCl and 17.5 µl of 1 M Tris pH 8.0.

Next, we repeated the elution treatment that was applied to the bisDRIP-seq samples. First, 0.5 µl of 100 U/µl RNase I was added to each sample. Samples were then incubated for 20 min at 50°C. RNAs still bound to DNA were degraded by adding 1 µl of 0.5 M MgCl₂ and 3 µl of 5 U/µl RNase H (New England Biolabs, Ipswich, Massachusetts) to each sample. The samples were then incubated for 20 min at 37°C. Next, we added 2 µl of 20 µg/µl of Proteinase K to each sample. The samples were then incubated for 1 hr at 50°C. Finally, the DNA in the sample was extracted using phenol-chloroform. After this point, the bisDRIP-seq protocol was followed as described above.

Both an RNase H-treated sample and matched control sample were prepared using a single plate of MCF-7 cells. These cells were treated as described in the initial steps of the bisDRIP-seq protocol described above. These initial steps included all steps until and including fragment ion of the DNA with restriction enzymes and confirmation of the digest with an agarose gel,

After those initial steps and immediately prior to immunoprecipitating RNA-DNA hybrids with S9.6 antibody, our sample was split into two samples. RNase H was added to one sample to a final concentration of 0.05 U/µl. We now call this sample the ‘RNase H-treated sample’. Dithiothreitol (at the same concentration as in the RNase H enzyme solution) was added to the other sample to a final concentration of 10 µM. RNase I was also added to both samples to a final concentration of 0.1 U/µl in order to prevent non-specific interactions between RNAs in solution and the S9.6 antibody (Phillips et al., 2013). Samples were then incubated for 2 hr at 37°C. Following this treatment, both samples were incubated with S9.6 antibody and Dynabeads Protein G as described above in the bisDRIP-seq protocol.

Next, we adjusted the immunoprecipitation wash steps to allow for further RNase H treatment. First, three wash steps were performed in immunoprecipitation buffer as described in the standard bisDRIP-seq protocol. Next, we added two additional wash steps using 200 µl of immunoprecipitation buffer supplemented with 3 mM MgCl₂. In each of these final two wash steps, RNase H was added to a final concentration of 0.5 U/µl to the ‘RNase H’ treated sample, while dithiothreitol was added to a final concentration of 50 µM to the matched control sample. Each wash lasted 20 min and during each wash the samples were mixed end over end at 37°C. On each occasion, the supernatant was removed after applying the sample to a magnet for 3 min.

After these wash steps, the samples were treated with NaOH and eluted as described in the bisDRIP-seq protocol above.

The delayed bisDRIP-seq sample was prepared largely following the bisDRIP-seq protocol, with a few modification.

After cells were washed in PBS as described in the bisDRIP-seq protocol, 5 ml of lysis buffer was added. This lysis buffer did not include bisulfite and was instead composed of 1% SDS, 50 mM Tris pH 8, and 10 mM EDTA. Next, 1 ml of 20 µg/µl Proteinase K was added to the sample. Samples were then incubated overnight at 37°C while being rotated end over end.

Next, nucleic acids were extracted using phenol-chloroform and ethanol precipitated as described in the bisDRIP-seq protocol.

At this point, we treated the nucleic acids with bisulfite. First, the nucleic acids were dissolved in 3.3 mM EDTA. Next, 900 µl of ammonium bisulfite solution and 26.8 µl hydroquinone were added to the sample. The sample was then incubated in a shaking incubator for 2.5 hr at 37°C at 1100 rpm, while being protected from light.

Next, the bisulfite was removed from the samples through dialysis. First, samples were added to 2.5 ml of buffer 3.1 lacking BSA (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl₂) in a 15 ml conical centrifuge tube. Samples were then added to an Amicon Ultra-4 Centrifugal Filter Unit with a 30,000 nominal molecular weight limit (EMD Millipore, Darmstadt, Germany). Next, the samples were centrifuged following the instructions of the centrifugal filter unit manufacturer. After centrifugation, fresh buffer 3.1 lacking BSA was added to the centrifugal filter unit and centrifugation was repeated. This dialysis process was repeated until the sample was nominally dialyzed at least 1000000 fold. At this point, the samples were resuspended in 850 µl of buffer 3.1 and the remaining steps of the bisDRIP-seq protocol were followed as described above.

Prior to constructing DNA sequencing libraries, the eluted DNA was fragmented into approximately 300 bp fragments. DNA from bisDRIP-seq reactions was re-suspended in 150 µl of 1XTE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were then fragmented using an S2-series Covaris ultrasonicator (Woburn, Massachusetts). Sonication was performed at 4°C using the following conditions: intensity 5, 10% Duty Intensity Factor, 200 cycles per burst and 140 s total treatment time.

We next used a Pico-Methyl Seq Library Prep Kit (Zymo Research, Irvine, California) to create the DNA sequencing library. This kit is primarily designed for mapping 5-methylcytosine in small quantities of genomic DNA. We omitted the bisulfite-treatment steps. Instead, we specifically followed the library preparation steps as instructed by the manufacturer. These steps amplified our DNA and used random priming to add indexes to our DNA library that are compatible with Illumina's TruSeq technology.

In preparation for sequencing, the quality of each DNA library was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) by the Weill Cornell Epigenomics Core (New York City, New York). The concentration of DNA was measured using a Quant-iT dsDNA Assay Kit and a SpectraMax M2 Microplate Reader following the instructions provided in the Quant-iT dsDNA Assay kit.

DNA libraries were sequenced by the Weill Cornell Epigenomics Core using a HiSeq 2500 System (Illumina, San Diego, California). Either five or six samples were loaded per lane. DNA libraries were sequenced following the manufacturer's instructions for single-index 100 bp paired-end read clustering.

Sequencing data was first processed using CASAVA 1.8.2 (Illumina, San Diego, California) to obtain the nucleotide sequence of reads in FASTQ format.

We next aligned reads to the GRCh38 reference genome. Reads were aligned to the genome using the Bismark software library version 0.14.3 Bismark tool (Krueger and Andrews, 2011). The Bismark software library is specifically designed to align bisulfite-modified reads. In this analysis, the Bismark library used the Bowtie2 alignment tool version 2.2.5 (Langmead and Salzberg, 2012) to perform the actual alignments. We also selected the Bismark option for post-bisulfite adapter tagging.

Read alignment was done in three phases. If we were unable to map a read in one phase, then we attempted to map the read in the following phase.

In the first alignment phase, full paired-end sequencing reads were aligned to the genome. First, reads were trimmed to remove both low confidence sequences and the very ends of reads. This was done using Flexbar version 2.5 (Dodt et al., 2012) with the following settings: quality format i1.8, phred minimum of 30, 10 nt removed from 5' end of reads. Next, the Bismark tool was used to align reads to the genome. The settings employed for the alignment did not allow for mismatched nucleotides and restricted the gap between paired-end reads to no more than 1000 bp.

Next, the Bismark alignment tool was repeated on all reads that failed to map in the first alignment phase. In this second phase, the two ends of paired-end reads were treated as single-end reads. The settings employed for alignment did not allow mismatched nucleotides.

In the final alignment phase, we attempted to align all reads that were not aligned to the genome in the first two phases after additional preprocessing. In this phase, we continued to treat both ends of paired-end reads as single-end reads. However, we began this phase by using Flexbar version 2.5 to remove all nucleotides 55 nt or further 3' of the start of the read. Reads were then aligned with the Bismark tool using settings that did not allow for mismatched nucleotides.

Having aligned reads to the genome, we next removed putative read duplicates. To remove read duplicates, we ran the Bismark deduplicate_bismark tool.

Finally, we determined if cytosines within aligned reads had been converted by bisulfite. To determine whether individual cytosines had been converted to uracils, we used the Bismark bismark_methylation_extractor tool. In the case of paired-end alignments, we used the –no_overlap setting. With single-end alignments, we used the --ignore_3prime 10 setting. This setting removed the final ten nucleotides of each alignment. These nucleotides were removed from aligned reads because we observed bias in the fraction of cytosines converted in this region of reads.

Next, we developed a method to score reads based on the likelihood that a given read was single-stranded in our samples. This measure was intended to filter out conversions that occur due to spontaneous breathing of DNA. Additionally, we wanted to ensure that single-stranded reads containing large numbers of cytosines were not given greater value than single-stranded reads containing small numbers of cytosines. This method was applied to on each sample separately.

For the purposes of calculating bisDRIP-seq scores, we define the ‘original number of cytosines’ as the sum of converted cytosines and unconverted cytosines.

The goal of this method was to estimate the fraction of reads that had a set number of conversions due to chance alone. The inverse fraction was then defined as the bisDRIP-seq score for all reads with that number of conversions. Therefore, all of the reads from the same sample, with the same original number of cytosines, and with the same number of converted cytosines were given the same bisDRIP-seq score.

First, we made a rough estimate of the background conversion rate for each sample. Initially, we calculated the total fraction of cytosines that were converted in our aligned reads. Next, we repeated this calculation after removing reads that had greater than 2.5 fold more conversions than the average across all reads. This value was used as an estimate of the background conversion rate.

Next, we calculated the probability of observing a specified number of converted cytosines in a given read by chance. This probability was calculated using the binomial distribution. In this binomial distribution calculation, the size of the sample was the original number of cytosines in the read and the probability of a conversion was the estimated background conversion rate.

Next, we calculated the number of reads expected to have a given original number of cytosines and a given number of converted cytosines. First, we calculated the total number of aligned reads with a given original number of cytosines. Next, we multiplied that number with the probability of observing the specified number of converted cytosines in a read with that original number of cytosines by chance.

Next, a bisDRIP-seq score was calculated for each read. First, reads were binned together based on the original number of cytosines in the read and the number of converted cytosines in the read. Next, bins were grouped together if they had the same original number of cytosines. If a group of reads with a given original number of cytosines per read consisted of more than a thousand reads in total and the expected number of reads for each bin was above five, then we calculated the bisDRIP-seq for each bin as:

This value was calculated first for reads with one original number of cytosines and then was calculated for reads with progressively higher numbers of original cytosines. If the expected number of reads for a bin was below five, then the bisDRIP-seq score was calculated for all reads with x number of converted cytosines using the equation:

In this equation, the original number of cytosines (n) is held constant.

If there were fewer than a thousand reads in a group, reads were scored differently. bisDRIP-seq scores with a specific number of conversions were given the same score as reads with the same number of conversions, but one fewer original number of cytosines. Given the total number of aligned reads per sample, the number of reads assigned scores on this basis was relatively small.

Source code for calculation of bisDRIP-seq scores from read sequence is in Source code 1 and processingbisDRIPseqreads.py, which has been deposited in https://github.com/champben2002/bisDRIPseq/

bisDRIP-seq scores were normalized to ensure that the sum of the bisDRIP-seq scores were the same across samples after normalization. This normalization procedure assumes that there are no global differences in the amount of single-stranded structure between samples. First, the sum of all read bisDRIP-seq scores was calculated for each sample. Next, the normalized bisDRIP-seq score for each read was calculated for each read in a given sample using the formula:

This normalization procedure was repeated for each sample separately.

All bisDRIP-seq scores in the manuscript used this standard normalization procedure, unless otherwise noted.

Promoter-associated R-loops have previously been associated with promoter regions enriched in guanines on the non-template strand (Ginno et al., 2012). We did not make conclusions related to this finding since bisDRIP-seq scores depend on the presence of cytosines in single-stranded regions of reads. However, we did consider whether our conclusions might be affected by the cytosine content of reads.

To test for whether our conclusions were affected by the cytosine content of reads, we applied a ‘cytosine normalization’ procedure to the raw bisDRIP-seq scores. First, reads within a sample were grouped based on the original number of cytosines in each read. Next, we normalized the reads within each group so that the average bisDRIP-seq score for each group would be identical. This normalization was done by multiplying the bisDRIP-seq score of each read within a group by a group-specific variable. Once this cytosine normalization was complete, the standard normalization procedure was followed as described above.

We repeated our analysis using these cytosine-content normalized scores (Figure 5—figure supplement 3). Our conclusions were not changed by this cytosine-content normalization.

Source code for bisDRIP-seq normalization is included in processingbisDRIPseqreads.py, which has been deposited in Source code 1 and https://github.com/champben2002/bisDRIPseq/

All genomic maps were generated using the Integrative Genomics Viewer version 2.3.59 (86) with the GRCh38 human genome (Robinson et al., 2011; Thorvaldsdóttir et al., 2013). The automatically loaded refseq gene models (O'Leary et al., 2016) were included in each map, with the following exceptions: For NEAT1, we used the gene model from GENCODE version 24 (Harrow et al., 2012) of the long isoform of NEAT1, ENST00000501122.2, since it appears more consistent with the RNA-seq data. For HIST1H2BK, we used the gene model from GENCODE version 24 (Harrow et al., 2012) of the intronless isoform of HIST1H2BK, ENST00000356950.1.

bisDRIP-seq scores were calculated for each nucleotide in the genome. The score of a given nucleotide in the genome was calculated as the sum of the bisDRIP-seq scores of reads that aligned to that nucleotide. Notably, this score was strand specific. The bisDRIP-seq score of reads was associated with a given nucleotide only if that nucleotide was on the same strand as the cytosines converted in the read. These bisDRIP-seq scores were then plotted for specific genomic loci.

Local sequence composition was plotted for sites in the HIST1H1E and HIST1H2BG genes. At each site, the mean number of adenines, cytosines, guanines and thymines in the surrounding region were plotted. The surrounding region included the nucleotide at the site, the twenty-five nucleotides 5' of the site and twenty-five nucleotides 3' of the site on the template strand.

bisDRIP-seq scores were calculated for genomic regions using the following procedure:

First, we summed the bisDRIP-seq scores of reads that completely aligned to the specified region.

Next, reads partially in the specified region were partially added to the specified region's score. The fraction of the read's score added to the score of the specified region's score was determined by the fraction of the read's length that aligned to the region. Thus the fraction of the read's length contained within the region was multiplied by the read's score. The product of this calculation was added to the specified region's score. This was repeated for each read that partially aligned in the specified region. This procedure provided the region's final bisDRIP-seq score.

In some of our analysis, bisDRIP-seq scores were calculated for only one strand within a specified region. In this case, the bisDRIP-seq scores from individual reads was only added to the specified region's bisDRIP-seq score if the read had the correct strand orientation. A read had the correct strand orientation if any converted cytosines in the read mapped to the correct strand.

Source code for calculating region scores is incorporated into regionbisDRIPseqscores.py, which has been deposited in Source code 2 and https://github.com/champben2002/bisDRIPseq/.

Monte Carlo simulations were used to determine the expected distribution of bisDRIP-seq scores if they distributed randomly across the genome. Two simulations were performed.

First, a simulation was performed in which reads were mapped to random regions across the genome. This simulates the possibility that both the RNA-DNA hybrid immunoprecipitation and the bisulfite mapping in bisDRIP-seq are non-specific. First, we split the genome into 1 kb regions. Next, we determined if any bisDRIP-seq reads aligned to a given 1 kb region. If no reads aligned to a given region, then that region was removed from further simulation steps. Reads were then associated with a random 1 kb region that was in the same chromosome to which they originally mapped. The region that each read was associated with was determined using the R 'random' package. This was repeated for each of our thirteen samples.

Second, a simulation was performed in which bisDRIP-seq scores were randomly shuffled between reads. This simulates the possibility that the bisDRIP-seq score associated with each read was stochastic. For each sample, we randomly shuffled all of the scores associated with reads aligned to a given chromosome. The random shuffle of read scores was performed using the R 'random' package. This simulation was repeated for each chromosome and for each of the thirteen bisDRIP-seq samples.

Next, bisDRIP-seq scores were calculated for each 1 kb region using either real bisDRIP-seq scores or using the products of one of the two simulations. This analysis used the same 1 kb regions discussed in the first Monte Carlo simulation.

Source code for Monte Carlo simulations are included in Monte_Carlo_random_assign_reads_to_regions.py and Monte_Carlo_for_shuffling_bisDRIPseq_scores.py, which have been deposited in Source code 3 and Source code 4 (respectively), as well as https://github.com/champben2002/bisDRIPseq/

GENCODE's annotated list of transcription start sites (Harrow et al., 2012) was used to generate a ‘reference GENCODE transcription start site list.’ This set of transcription start sites was then used to define promoter regions in the genome and as reference points for metaplots.

First, the GENCODE comprehensive gene annotation release 25 was downloaded from GENCODE's website.

Next, transcription start sites were removed from the transcription start site list if they were within a thousand base pairs of a higher priority transcription start site. The priority of transcription start sites was determined as follows:

1. The highest priority transcription start sites were transcription start sites within 25 bp of a site identified by CAGE-seq. Transcription start sites near sites that had higher CAGE-seq signal were given a higher priority than transcription start sites near sites with lower CAGE-seq signal.

2. Priority was next determined by the confidence annotation scores associated with each transcription start site in the GENCODE annotations.

3. Finally, priority was established using a random number generated using the Python ‘random’ package.

After removing lower priority transcription start sites, we had a final reference GENCODE transcription start site list. Promoter regions were then defined as the region within a thousand base pairs of each transcription start site in our reference GENCODE transcription start site list. See Source_data_file_1.xls for all transcription start sites in this final list

In later analysis, inactive promoter regions were removed. Unless noted otherwise, inactive promoter regions were defined as promoter regions with sense-strand promoter activity in the bottom eighty percentile between the transcription start site and + 1000 bp.

In order to calculate the promoter activity of each promoter, we used publicly available GRO-seq data obtained from MCF-7 cells by Hah et al., 2013. In particular, data was combined from files GSM1067410, GSM1067411, GSM1067412, GSM1067413, GSM1067414 and GSM1067415. The MCF-7 cells used in these samples were treated in a similar manner to the MCF-7 cells used in our bisDRIP-seq protocols.

Unless noted otherwise, promoter activity was defined by the mean number of sense-strand GRO-seq reads measured in the region between the transcription start site and + 1000 bp. The method for calculating the number of reads in this region of each promoter was similar to method used to calculate bisDRIP-seq scores for regions.

For each promoter region, we first summed the number of GRO-seq reads that completely aligned to the region between the transcription start site and + 1000 bp.

Next, reads partially in the specified region were partially added to the specified region's score. The fraction of the read's length contained within the region between the transcription start site and + 1000 bp was added to the promoter region's score. This was repeated for each read that partially aligned to the promoter region. This procedure provided the promoter region's final promoter activity.

A similar procedure was followed to determine the antisense promoter activity and the promoter activity between the transcription start site and + 250 bp.

Exon-intron junctions were extracted from the GENCODE comprehensive gene annotation release 25 (Harrow et al., 2012). Exon-intron and intron-exon junctions were separated based on whether they were the first exon-intron, first intron-exon or second exon-intron junction in a given annotated transcript. If two promoter regions shared the same exon-intron junction, then one was removed from the list.

Next, all exon-intron junctions were removed that were associated with inactive promoter regions. Inactive promoter regions did not contain positive R-loop signal in Figure 3F, suggesting that removing these promoter regions would lead to a greater signal-to-noise ratio. The activity of promoter regions was defined here based on the number of sense-strand GRO-seq reads that aligned to the promoter region between the transcription start site and + 1000 bp. Promoters in the bottom eighty percentile in terms of promoter activity were considered inactive promoters and removed from exon-intron junction analysis.

Exon-intron junctions associated with the intronless genes, MALAT1 and NEAT1, were also removed.

This procedure provided a final reference set of exon-intron junctions. See Figure 4—source data 1 for this final reference list.

First-exon length was calculated for each first exon-intron junction in the final reference set of first exon-intron junctions. The first-exon length was calculated as the distance between the transcription start site and the first exon-intron junction. This list of first-exon lengths was used to calculate the median length of all first exons for active promoter regions.

In order to compare some external dataset to our data, it was necessary to convert the coordinates of sites or regions to the GRCh38 genome from an earlier version of the reference human genome. In all cases, this was accomplished using the UCSC utility liftOver (Speir et al., 2016).

Cap analysis gene expression sequencing (CAGE-seq) data came from the publicly-available ENCODE MCF-7 dataset ENCFF207DXM (ENCODE Project Consortium, 2012).

In order to compare bisDRIP-seq datasets with DRIPc-seq datasets, we used the DRIPc-seq data of Sanz et al., 2016. In particular, we compared our data against the data deposited in the Gene Expression Omnibus GSM1720613 and GSM1720614 wig files.

In order to compare bisDRIP-seq datasets with DRIP-seq datasets, we used the DRIP-seq data of Sanz et al., 2016. In particular, we compared our data against the data deposited in the Gene Expression Omnibus GSM1720615 and GSM1720616 wig files.

Total cell RNA-seq data displayed for individual genes came from ENCODE files ENCFF426WXY and ENCFF866OVQ (ENCODE Project Consortium, 2012). The RNA-seq experiments used to generate that data were rRNA depleted and used MCF-7 cells.

Nucleus RNA-seq levels of active genes in Figure 5—figure supplement 1A are the mean transcripts per kilobase million (TPM) values as measured in ENCODE files ENCFF063BLU and ENCFF285GOS (ENCODE Project Consortium, 2012). These RNA-seq experiments sequenced mRNA depleted in rRNA from the nuclei of MCF-7 cells.

RNA Polymerase II levels were calculated using the ENCODE files ENCFF496YAE and ENCFF881YOO (ENCODE Project Consortium, 2012). These datasets comes from chromatin immunoprecipitations of POLR2A in MCF-7 cells. The number of reads between the transcription start site and + 250 bp of each gene was calculated using the same methodology described above for GRO-seq reads.

For DRIP-seq and bisDRIP-seq promoter region enrichment tests, only promoter regions in genes larger than 2 kb were considered. For each promoter region, a matched region was selected from the same gene. This matched region was centered on an exonic site more than 2 kb from the transcription start site. Promoter enrichment was then tested using the non-parametric Wilcoxon signed-rank test described below.

Promoter region ranking was only performed on active promoter regions. Promoter activity was defined here based on the number of sense-strand GRO-seq reads that aligned to the promoter region between the transcription start site and +250 bp. Active promoter regions were promoter regions in the top twenty percentile of promoter regions in terms of promoter activity. This filter was intended to filter out inactive promoter regions, which appeared to only contribute noise to our analysis.

Next, promoter regions were filtered on the basis of sense-strand bisDRIP-seq scores. The non-template strand bisDRIP-seq score had to be positive in the region between the transcription start site and + 250 bp in all control-treated biological replicates. Otherwise, the promoter region was removed from out list.

The remaining promoter regions were scored based on the value:

2 x (log2(control-treated non-template bisDRIP-seq score + 1) - log2(triptolide-treated non-template bisDRIP-seq score + 1)) - log2(control-treated template bisDRIP-seq score + 1))

Loess smoothed plots were generated using the r fANCOVA package's loess.as algorithm with parameters ‘criterion = aicc’ and ‘family = gaussian’.

Scatterplots were generated with the ggplot2 package geom_points() algorithm, using the lm option to plot a linear regression line of the displayed points.

Violin plots and jitter plots were created using the ggplot2 package. Jitter plots were created using geom_jitter() with height = 0, width = 0.725. Violin plots were created using geom_violin() with alpha = 0.5.

bisDRIP-seq score metaplots were created in relation to reference points. These reference points were either transcription start sites or exon-intron junctions, depending on the analysis. The final metaplot associated a bisDRIP-seq score to each ‘location’ relative to a given reference point. Location refers to the distance from the reference point and whether the site is upstream or downstream of the reference site. For a given read, we determined the location of each nucleotide in the read relative to the proximal reference point. Next, the location of each nucleotide was associated with the bisDRIP-seq score of the read. After repeating this process for all reads, all of the scores associated with a given location were summed unless otherwise noted. This sum was then used as the bisDRIP-seq score for that location in the metaplot. Finally, each location was plotted against the bisDRIP-seq score associated with that location.

Source code for metaplot analysis is incorporated into bisDRIPseqmetaplotanalysis.py, which has been deposited in Source code 5 and https://github.com/champben2002/bisDRIPseq/.

The conversion asymmetry metaplot was generated in relation to transcription start sites. The total number of conversions was summed at each site relative to the transcription start site across all active promoter regions. This was repeated for each sample, strand and sample type. Next, we calculated the mean number of conversions across samples for each position relative to the transcription start site. Next, we subtracted the mean number of conversions for each position relative to the transcription start site in triptolide-treated samples from the number of conversions observed in control-treated samples. Finally, we plotted the log ratio of the tripolide-corrected number of conversions on the non-template strand to the tripolide-corrected number of conversions on the template strand.

To create a metaplot of the percentage of cytosines converted on the template strand, we calculated the number of cytosines and converted cytosines that aligned to a given site relative to the transcription start site. Next, the number of converted cytosines was divided by the total number of original cytosines that aligned to that site.

DRIPc-seq metaplots were generated using essentially the same process as was used for bisDRIP-seq score. Instead of using the scores of reads, we used the read-count associated with regions in the wig file.

In all plots of R-loop signal, R-loop signal = (control-treated sample, non-template bisDRIP-seq score - triptolide-treated sample, non-template bisDRIP-seq score) - (control-treated sample, template bisDRIP-seq score - triptolide-treated sample, template bisDRIP-seq score).

In all plots where a log2 transformation was applied to a dataset, each value in the dataset was added to one prior to the log2 transformation.

To sample the reads from the MALAT1 R-loop forming region, we selected all reads that: (1) aligned to the non-template strand of MALAT1, (2) start and end between the transcription start site and + 1600 bp, and (3) are larger than 75 bp long. Next, we randomly selected twenty-five reads. Reads were then plotted using ggplot2's geom_point function.

Since we are not confident that our data follows a Gaussian distribution, we typically used non-parametric tests of significance. In particular, we applied the Wilcoxon signed-rank test whether various datasets were significantly different. Wilcoxon signed-rank tests were performed using the two-sided R wilcox.test() algorithm using default parameters with the exception of the ‘paired’ setting which was set to ‘FALSE’.

In the case of comparisons between metaplots, a conservative multiple-hypothesis correction was performed on the p-values derived from Wilcoxon signed-rank tests. This multiple-hypothesis correction involved multiplying the p-value of an individual nucleotide position by 2001, since the metaplots involved examining 2001 nucleotide positions simultaneously.

The significance of correlations between datasets was examined using the Spearman's rank-correlation test with asymptotic t approximation. Spearman's rank-correlation tests were used since we are not confident that our data follows a Gaussian distribution and therefore wished to apply a non-parametric test of correlation. Spearman's rank-correlation tests were performed using the R cor.test(x, y, method = ‘spearman’) algorithm.

For each sample, we deposited the sequence reads, the conversion frequency at each cytosine nucleotide and a bedGraph of bisDRIP-seq scores in GEO series GSE98886: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE98886

Private token for reviewers: azyfacgczpertip

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank C Kam and members of the Jaffrey laboratory for comments and suggestions, the members of the Weill Cornell Epigenomics Core for their assistance in high-throughput sequencing, and the Mittal lab (Weill-Cornell) for their assistance with DNA sonication. This work was supported by a Tri-Institutional Starr Stem Cell Scholars Fellowship (JD) and NIH grant R01 CA186702 (SRJ).

1. Received: May 3, 2017
2. Accepted: October 22, 2017
3. Accepted Manuscript published: October 26, 2017 (version 1)
4. Version of Record published: November 28, 2017 (version 2)

© 2017, Dumelie et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.