Fig. 2

Genome-wide detection of R-loops in yeast by ssDRIP-seq. (A) Diagram of ssDRIP-seq in yeast under vegetative growth conditions. Nuclei were isolated from yeast cells by grinding well in liquid nitrogen, followed by genome DNA (gDNA) extraction. gDNA was fragmented using endonucleases, and DNA:RNA hybrids were captured by using the S9.6 antibody. The DRIPed DNA samples were ligated to the adapter on the 3’ end of the ssDNA using Adaptase. The extension step was performed using the primer paired to the first adapter, followed by a ligation reaction to add the second truncated adapter. An indexing PCR step was performed to add the indexed sequence, and the library was amplified and sequenced. (B) Pairwise comparison of two ssDRIP-seq replicates in mitosis. The Pearson correlation coefficient was computed from each comparison to evaluate the reproducibility. RNase H treatment served as a negative control. (C) Snapshot of the ssDRIP-seq data in yeast under vegetative growth conditions and RNase H treatment, including R-loop (grey), wR-loop (blue) and cR-loop (red) in a representative genomic region (chrII:401,500–827,934). (D) Overlap of peaks identified by wR-loop and cR-loop based on peak count in yeast under vegetative growth conditions. (E) The size distribution of R-loop (red), wR-loop (yellow) and cR-loop (green) peaks in yeast under vegetative growth conditions determined by the peak calling strategies of MACS2 with default settings to call narrow R-loop peaks [63]. (F) DNA motif in the peak regions of unstranded R-loops, wR-loops and cR-loops in yeast under vegetative growth conditions that were identified by MEME-ChIP. E-values are provided on the right. (G) The genomic distribution of ssDRIP-seq mapped R-loops, wR-loops and cR-loops that were identified by ChIPseeker. Various genomic regions are colour coded according to the labels on the bottom. Blue, promoter; green, 1st exon; yellow, other exon; black, intron; orange, downstream [ ≤ = 300 bp]; red, distal intergenic [> 500 bp from TSS and > 300 bp from TES]. (H) Heatmap of RNAPII signals in regions ± 3 kb from the R-loop on the genomes of yeast cells. The RNAPII data are from RNAPII ChIP sequencing from Morselli et al. [64]