Supplementary Figure 5: R-loop accumulation on repeat elements (REs).
(a) RNA:DNA hybrids were detected on dot plots of genomic DNA isolated from the indicated genotypes grown at 20 °C or 25 °C (three blots at each temperature were quantified by scanning for Fig. 5a). Equal amounts of DNA (determined by OD260/280) extracted from adult worms were spotted with or without RNase H treatment in decreasing concentrations (4, 2 and 1 μg). The nitrocellulose membrane was probed with the RNA:DNA-specific S9.6 antibody (n(20 °C) = 3, n(25 °C) = 3). Quantification on the right side with signals normalized to the background of each blot. (b) DRIP-seq signals in wt embryos are shown for genes grouped on the basis of their transcriptional activity. The upper box blot shows the level of transcription of the separate groups (N = 1). This enhancement of R loops on very highly expressed genes has been observed in many organisms. (c,d) Graphs show the accumulation of RNA:DNA hybrids in wt and met-2 set-25 embryos relative to the distribution of H3K9me2 and me3 over the rDNA cluster (c) or the right telomere of chr. I (d), in wt embryos. (e) DRIP-seq examples showing the R-loop signal over two RE clusters. The ChIP signal from antibody S9.6, which is specific for RNA:DNA hybrids, was normalized to input, and the RNase H control values were subtracted.
